Electronic methods for the detection of analytes

ABSTRACT

The present invention is directed to the detection of target analytes using electronic techniques, particularly AC techniques.

CROSS-REFERENCED TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 12/773,382,filed May 4, 2010, now U.S. Pat. No. 8,114,661, which is a continuationof U.S. patent application Ser. No. 12/366,471, filed Feb. 5, 2009, nowU.S. Pat. No. 7,759,073, which is a continuation of U.S. patentapplication Ser. No. 09/096,593, filed Jun. 12, 1998, now U.S. Pat. No.7,560,237, which claims benefit of U.S. Patent Application No.60/049,489, filed Jun. 12, 1997, now expired, which allapplication/patents are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The invention relates to analytical methods and apparatus, andparticularly to the detection of analytes, including biomolecules, usingelectronic techniques, particularly AC techniques.

BACKGROUND OF THE INVENTION

There are a number of assays and sensors for the detection of thepresence and/or concentration of specific substances in fluids andgases. Many of these rely on specific ligand/antiligand reactions as themechanism of detection. That is, pairs of substances (i.e. the bindingpairs or ligand/antiligands) are known to bind to each other, whilebinding little or not at all to other substances. This has been thefocus of a number of techniques that utilize these binding pairs for thedetection of the complexes. These generally are done by labeling onecomponent of the complex in some way, so as to make the entire complexdetectable, using, for example, radioisotopes, fluorescent and otheroptically active molecules, enzymes, etc.

Other assays rely on electronic signals for detection. Of particularinterest are biosensors. At least two types of biosensors are known;enzyme-based or metabolic biosensors and binding or bioaffinity sensors.See for example U.S. Pat. Nos. 4,713,347; 5,192,507; 4,920,047;3,873,267; and references disclosed therein. While some of these knownsensors use alternating current (AC) techniques, these techniques aregenerally limited to the detection of differences in bulk (ordielectric) impedance, and rely on the use of mediators in solution toshuttle the charge to the electrode.

Recently, there have been several preliminary reports on the use of veryshort connections between a binding ligand and the electrode, for directdetection, i.e. without the use of mediators. See Lotzbeyer et al.,Bioelectrochemistry and Bioenergetics 42:1-6 (1997); Dong et al.,Bioelectrochemistry and Bioenergetics 42:7-13 (1997).

In addition, there are a number of reports of self-assembled monolayersof conjugated oligomers on surfaces such as gold. See for example Cyganet al., J. Am. Chem. Soc. 120:2721 (1998).

In addition, Charych et al. report on the direct colormetric detectionof a receptor-ligand interaction using a bilayer assembly (Science261:585 (1993).

Accordingly, it is an object of the invention to provide novel methodsand compositions for the detection of target analytes using ACtechniques.

SUMMARY OF THE INVENTION

Accordingly, in accordance with the above objects, the present inventionprovides methods of detecting a target analyte in a test samplecomprising a redox active molecule and an analyte The method comprisesapplying an input signal to the test sample and detecting a change inthe faradaic impedance of the system as a result of the association ofthe redox active molecule with the analyte.

In an additional aspect, the invention provides methods binding thetarget analyte to a redox active complex comprising a redox activemolecule and a binding ligand which will bind the target analyte,followed by detection of a change in the faradaic impedance of thesystem as a result of the association of the redox active molecule withthe target analyte, if present.

The methods further comprising applying a first input signal to saidredox active complex; the input signal can comprise an AC componentand/or a DC component.

In a further aspect, the invention provides apparatus for the detectionof analyte in a test sample, comprising a test chamber comprising atleast a first and a second measuring electrode, wherein the firstmeasuring electrode comprises a covalently attached ligand for ananalyte, and an AC/DC voltage source electrically connected to the testchamber.

In an additional aspect, the present invention provides metal ionsensors comprising electrodes comprising self-assembled monolayers andat least one metal ion ligand or chelate covalently attached to theelectrode via a conductive oligomer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the synthetic scheme for a conductive polymer containingan aromatic group with a substitution group. The conductive oligomer isa phenyl-acetylene Structure 5 oligomer with a single methyl R group oneach phenyl ring, although other oligomers may be used, and terminatesin an ethyl pyridine protecting group, as described herein, forattachment to gold electrodes.

FIG. 2 depicts the synthetic scheme for making a redox active complexcomprising a RAM, in this case ferrocene, to a protein binding ligand,in this case an antibody, using a standard coupling reaction. As will beappreciated by those in the art, any proteins that either containsuitable amines or can be derivatized to contain a suitable amine can beadded in this manner. Alternatively, the amine may be added to the RAMand the BL contains the carboxylic acid. Similarly, while this figuredepicts the attachment of a RAM (ferrocene), a similar reaction may bedone with a conductive oligomer terminating in a carboxylic acid (or anamine), for attachment to a proteinaceous binding ligand. In addition,while not depicted, “Z” linkers, as described below, may be added inbetween the components.

FIG. 3 depicts the synthetic scheme of a System 3 type sensor,comprising a conductive oligomer containing a redox active complex of aRAM (in this case ferrocene), with a binding ligand (in this case abiotin derivative). Any number of other RAMs and BLs may be used.Additional “Z” linkers, as described below, may be added in between thecomponents. As for FIG. 2, a standard coupling agent (carbodimide) isused, which allows the attachment of virtually any amine- or carboxylicacid-containing moieties. The first subunit of the conductive oligomeris used, and then subsequent subunits are added.

FIG. 4 depicts a sensor of the invention, directed to the detection ofantibodies to a drug. Upon the introduction of a patient sample, withbinding of the antibody to its antigen, the environment of the RAM isaltered, leading to a detectable change in the signal (i.e. analteration in the faradaic impedance). The redox potential of the RAMmay be altered, or there may be a signal increase or alteration. As willbe appreciated by those in the art, the drug in this case could bereplaced by virtually any binding ligand. In addition, this type ofreaction may be run as a standard competitive type assay.

FIGS. 5A, 5B and 5C depict some possible ion sensors. The binding ofions such as Li+, Mg+2 or Na+ can alter the redox potential of theferrocene by altering the electron withdrawing properties of the crownethers, thus effecting a change in the signal upon binding.

FIGS. 6A, 6B, 6C, 6D and 6E depict a metal ion sensor embodiment of theinvention. FIG. 6A depicts a chelate metal ion binding ligand, in thiscase phenanthroline, that was subsequently attached to a gold electrode,with a monolayer present. FIGS. 6B and 6C depict AC scans in the absence(6B) and presence (6C) of FeCl₂, showing a peak around 450 mV, the redoxpotential of the iron. FIG. 6D depicts the same composition in thepresence of Ru(NH₃)₄PyCl, with a peak at around 650 mV. FIG. 6E depictsthe same composition in the presence of K_(I)Fe^(II)(CN)₆, also with apeak around 650 mV.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to the detection of analytes usingalternating current (AC) (also sometimes referred to as alternatingvoltage (AV)) techniques. The invention is based on the fact that atleast one redox property of a redox active molecule may be altered as aresult of its association with a target analyte. Without being bound bytheory, it appears that changes in the environment of the redox activemolecule can result in altered redox properties. That is, uponassociation of the analyte and the redox active molecule in some way, ameasurable redox property of the redox active molecule changes, thusallowing the detection of the analyte. In particular, it has beendiscovered that relatively small changes in measurable redox propertiescan be detected using AC techniques, enabling a variety of possiblebiosensors.

The change in a redox property of the redox active molecule is a resultof the association with an analyte. This may be due to a binding event,which may alter the conformation or accessibility of the redox activemolecule, and/or a change in the local environment of the redox activemolecule (for example in the solvent reorganization energy), both ofwhich will alter the faradaic impedance of the system, which in turnresults in a characteristic output signal, i.e. a different outputsignal than is received in the absence of the target analyte.

Accordingly, the present invention is directed to the detection ofanalytes using changes in the faradaic impedance of the system as aresult of the binding or association of an analyte. By “faradaicimpedance” herein is meant the impedance between the redox activemolecule and the electrode. Changes in capacitance (e.g. due to bindingof compounds to the surface or bulk dielectric capacitance) are notincluded in the definition of changes in faradaic impedance. This isquite different from the bulk or dielectric impedance, which is theimpedance of the bulk solution between the electrodes. Many factors maychange the faradaic impedance which may not effect the bulk impedance,and vice versa. As described herein, any number of perturbations of thesystem can result in an altered faradaic impedance, which may then serveas the basis of an assay. These include, but are not limited to, changesin electronic coupling of the redox active molecule and the electrode(often referred to as H_(AB) in the literature); changes in λ, thenuclear reorganization energy, which is usually dominated by the solventreorganization energy; changes in E₀ of the redox active molecule; thecharge transfer impedance of the redox active molecule in the system;the mass transfer impedance of the redox active molecule in the system;changes in the redox active molecule, including exchange of ligands ormetal ions; etc.

Systems relying on changes in faradaic impedance can be distinguishedfrom prior art systems on the basis of the use of mediators. That is,prior art systems usually rely on the use of soluble mediators toshuttle electrons between the redox active molecules and the electrode;however, the present invention relies on direct electron transferbetween the redox active molecule and the electrode, generally throughthe use of conductive oligomers. Thus, the methods of the presentinvention are generally run in the absence of soluble mediators thatserve to electronically mediate the redox active molecule and theelectrode. That is, the redox active molecules (RAMs) are directlyattached to the electrodes of the invention, rather than relying on bulkdiffusion mechanisms. Mediators in this context are to be distinguishedby co-reductants and co-oxidants, as generally described below.

Generally, compositions and methods described in PCT US97/20014, herebyexplicitly incorporated herein by reference in its entirety, find use inthe present invention.

Thus the present invention is directed to methods and compositions forthe detection of target analytes in test samples. By “target analyte” or“analyte” or grammatical equivalents herein is meant any molecule,compound or particle to be detected. As outlined below, target analytespreferably bind to binding ligands, as is more fully described below. Aswill be appreciated by those in the art, a large number of analytes maybe detected using the present methods; basically, any target analyte forwhich a binding ligand, described below, may be made may be detectedusing the methods of the invention.

Suitable analytes include organic and inorganic molecules, includingbiomolecules. In a preferred embodiment, the analyte may be anenvironmental pollutant (including pesticides, insecticides, toxins,etc.); a chemical (including solvents, polymers, organic materials,etc.); therapeutic molecules (including therapeutic and abused drugs,antibiotics, etc.); biomolecules (including hormones, cytokines,proteins, lipids, carbohydrates, cellular membrane antigens andreceptors (neural, hormonal, nutrient, and cell surface receptors) ortheir ligands, etc); whole cells (including procaryotic (such aspathogenic bacteria) and eukaryotic cells, including mammalian tumorcells); viruses (including retroviruses, herpesviruses, adenoviruses,lentiviruses, etc.); and spores; etc. Particularly preferred analytesare environmental pollutants; nucleic acids; proteins (includingenzymes, antibodies, antigens, growth factors, cytokines, etc);therapeutic and abused drugs; cells; and viruses.

In a preferred embodiment, the target analytes are not nucleic acids.Similarly, a preferred embodiment utilizes target analytes that are notglucose, and redox active complexes that do not contain glucose oxidase.

In a preferred embodiment, the target analyte is a protein. As will beappreciated by those in the art, there are a large number of possibleproteinaceous target analytes that may be detected using the presentinvention. By “proteins” or grammatical equivalents herein is meantproteins, oligopeptides and peptides, derivatives and analogs, includingproteins containing non-naturally occurring amino acids and amino acidanalogs, and peptidomimetic structures. The side chains may be in eitherthe (R) or the (S) configuration. In a preferred embodiment, the aminoacids are in the (S) or L-configuration. As discussed below, when theprotein is used as a binding ligand, it may be desirable to utilizeprotein analogs to retard degradation by sample contaminants.

Suitable protein target analytes include, but are not limited to, (1)immunoglobulins, particularly IgEs, IgGs and IgMs, and particularlytherapeutically or diagnostically relevant antibodies, including but notlimited to, for example, antibodies to human albumin, apolipoproteins(including apolipoprotein E), human chorionic gonadotropin, cortisol,α-fetoprotein, thyroxin, thyroid stimulating hormone (TSH),antithrombin, antibodies to pharmaceuticals (including antieptilepticdrugs (phenyloin, primidone, carbariezepin, ethosuximide, valproic acid,and phenobarbitol), cardioactive drugs (digoxin, lidocaine,procainamide, and disopyramide), bronchodilators (theophylline),antibiotics (chloramphenicol, sulfonamides), antidepressants,immunosuppresants, abused drugs (amphetamine, methamphetamine,cannabinoids, cocaine and opiates) and antibodies to any number ofviruses (including orthomyxoviruses, (e.g. influenza virus),paramyxoviruses (e.g respiratory syncytial virus, mumps virus, measlesvirus), adenoviruses, rhinoviruses, coronaviruses, reoviruses,togaviruses (e.g. rubella virus), parvoviruses, poxviruses (e.g. variolavirus, vaccinia virus), enteroviruses (e.g. poliovirus, coxsackievirus),hepatitis viruses (including A, B and C), herpesviruses (e.g. Herpessimplex virus, varicella-zoster virus, cytomegalovirus, Epstein-Barrvirus), rotaviruses, Norwalk viruses, hantavirus, arenavirus,rhabdovirus (e.g. rabies virus), retroviruses (including HIV, HTLV-I and-II), papovaviruses (e.g. papillomavirus), polyomaviruses, andpicornaviruses, and the like), and bacteria (including a wide variety ofpathogenic and non-pathogenic prokaryotes of interest includingBacillus; Vibrio, e.g. V. cholerae; Escherichia, e.g. Enterotoxigenic E.coli, Shigella, e.g. S. dysenteriae; Salmonella, e.g. S. typhi;Mycobacterium e.g. M. tuberculosis, M. leprae; Clostridium, e.g. C.botulinum, C. tetani, C. difficile, C. perfringens; Cornyebacterium,e.g. C. diphtheriae; Streptococcus, S. pyogenes, S. pneumoniae;Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H. influenzae;Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia, e.g. G.lamblia Y. pestis, Pseudomonas, e.g. P. aeruginosa, P. putida;Chlamydia, e.g. C. trachomatis; Bordetella, e.g. B. pertussis;Treponema, e.g. T. palladium; and the like); (2) enzymes (and otherproteins), including but not limited to, enzymes used as indicators ofor treatment for heart disease, including creatine kinase, lactatedehydrogenase, aspartate amino transferase, troponin T, myoglobin,fibrinogen, cholesterol, triglycerides, thrombin, tissue plasminogenactivator (tPA); pancreatic disease indicators including amylase,lipase, chymotrypsin and trypsin; liver function enzymes and proteinsincluding cholinesterase, bilirubin, and alkaline phosphotase; aldolase,prostatic acid phosphatase, terminal deoxynucleotidyl transferase, andbacterial and viral enzymes such as HIV protease; (3) hormones andcytokines (many of which serve as ligands for cellular receptors) suchas erythropoietin (EPO), thrombopoietin (TPO), the interleukins(including IL-1 through IL-17), insulin, insulin-like growth factors(including IGF-1 and -2), epidermal growth factor (EGF), transforminggrowth factors (including TGF-α and TGF-β), human growth hormone,transferrin, epidermal growth factor (EGF), low density lipoprotein,high density lipoprotein, leptin, VEGF, PDGF, ciliary neurotrophicfactor, prolactin, adrenocorticotropic hormone (ACTH), calcitonin, humanchorionic gonadotropin, cotrisol, estradiol, follicle stimulatinghormone (FSH), thyroid-stimulating hormone (TSH), leutinzing hormone(LH), progeterone, testosterone; and (4) other proteins (includingα-fetoprotein, carcinoembryonic antigen CEA.

In addition, any of the biomolecules for which antibodies may bedetected may be detected directly as well; that is, detection of virusor bacterial cells, therapeutic and abused drugs, etc., may be donedirectly.

Suitable target analytes include carbohydrates, including but notlimited to, markers for breast cancer (CA15-3, CA 549, CA 27.29),mucin-like carcinoma associated antigen (MCA), ovarian cancer (CA125),pancreatic cancer (DE-PAN-2), and colorectal and pancreatic cancer (CA19, CA 50, CA242).

Suitable target analytes include metal ions, particularly heavy and/ortoxic metals, including but not limited to, aluminum, arsenic, cadmium,selenium, cobalt, copper, chromium, lead, silver and nickel.

In a preferred embodiment, the target analyte is added to or introducedto a redox active molecule or redox active complex. By “redox activemolecule” or “RAM” or “electron transfer moiety” or “ETM” herein ismeant a compound which is capable of reversibly, semi-reversibly, orirreversibly transferring one or more electrons. The terms “electrondonor moiety”, “electron acceptor moiety”, and “electron transfermoieties” or grammatical equivalents herein refers to molecules capableof electron transfer under certain conditions. It is to be understoodthat electron donor and acceptor capabilities are relative; that is, amolecule which can lose an electron under certain experimentalconditions will be able to accept an electron under differentexperimental conditions. It is to be understood that the number ofpossible electron donor moieties and electron acceptor moieties is verylarge, and that one skilled in the art of electron transfer compoundswill be able to utilize a number of compounds in the present invention.Preferred electron transfer moieties include, but are not limited to,transition metal complexes, organic electron transfer moieties, andelectrodes.

In a preferred embodiment, the electron transfer moieties are transitionmetal complexes. Transition metals include those whose atoms have apartial or complete d shell of electrons; elements having the atomicnumbers 21-30, 39-48, 57-80 and the lanthanide series. Suitabletransition metals for use in the invention include, but are not limitedto, cadmium (Cd), copper (Cu), cobalt (Co), palladium (Pd), zinc (Zn),iron (Fe), ruthenium (Ru), rhodium (Rh), osmium (Os), rhenium (Re),platinium (Pt), scandium (Sc), titanium (Ti), Vanadium (V), chromium(Cr), manganese (Mn), nickel (Ni), Molybdenum (Mo), technetium (Tc),tungsten (W), and iridium (Ir). That is, the first series of transitionmetals, the platinum metals (Ru, Rh, Pd, Os, Ir and Pt), along with Fe,Re, W, Mo and Tc, are preferred. Particularly preferred are ruthenium,rhenium, osmium, platinium, cobalt and iron.

The transition metals are complexed with a variety of ligands, generallydepicted herein as “L”, to form suitable transition metal complexes, asis well known in the art. Suitable ligands fall into two categories:ligands which use nitrogen, oxygen, sulfur, carbon or phosphorus atoms(depending on the metal ion) as the coordination atoms (generallyreferred to in the literature as sigma (a) donors) and organometallicligands such as metallocene ligands (generally referred to in theliterature as pi (π) donors, and depicted herein as L_(m)). Suitablenitrogen donating ligands are well known in the art and include, but arenot limited to, NH₂; NHR; NRR′; pyridine; pyrazine; isonicotinamide;imidazole; bipyridine and substituted derivatives of bipyridine;terpyridine and substituted derivatives; phenanthrolines, particularly1,10-phenanthroline (abbreviated phen) and substituted derivatives ofphenanthrolines such as 4,7-dimethylphenanthroline anddipyridol[3,2-a:2′,3′-c]phenazine (abbreviated dppz); dipyridophenazine;1,4,5,8,9,12-hexaazatriphenylene (abbreviated hat);9,10-phenanthrenequinone diimine (abbreviated phi);1,4,5,8-tetraazaphenanthrene (abbreviated tap);1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam) and isocyanide.Substituted derivatives, including fused derivatives, may also be used.In some embodiments, porphyrins and substituted derivatives of theporphyrin family may be used. See for example, ComprehensiveCoordination Chemistry, Ed. Wilkinson et al., Pergammon Press, 1987,Chapters 13.2 (pp 73-98), 21.1 (pp. 813-898) and 21.3 (pp 915-957), allof which are hereby expressly incorporated by reference.

Suitable sigma donating ligands using carbon, oxygen, sulfur andphosphorus are known in the art. For example, suitable sigma carbondonors are found in Cotton and Wilkenson, Advanced Organic Chemistry,5th Edition, John Wiley & Sons, 1988, hereby incorporated by reference;see page 38, for example. Similarly, suitable oxygen ligands includecrown ethers, water and others known in the art. Phosphines andsubstituted phosphines are also suitable; see page 38 of Cotton andWilkenson.

The oxygen, sulfur, phosphorus and nitrogen-donating ligands areattached in such a manner as to allow the heteroatoms to serve ascoordination atoms.

In a preferred embodiment, organometallic ligands are used. In additionto purely organic compounds for use as redox moieties, and varioustransition metal coordination complexes with & bonded organic ligandwith donor atoms as heterocyclic or exocyclic substituents, there isavailable a wide variety of transition metal organometallic compoundswith π-bonded organic ligands (see Advanced Inorganic Chemistry, 5thEd., Cotton & Wilkinson, John Wiley & Sons, 1988, chapter 26;Organometallics, A Concise Introduction, Elschenbroich et al., 2nd Ed.,1992, VCH; and Comprehensive Organometallic Chemistry II, A Review ofthe Literature 1982-1994, Abel et al. Ed., Vol. 7, chapters 7, 8, 10 &11, Pergamon Press, hereby expressly incorporated by reference). Suchorganometallic ligands include cyclic aromatic compounds such as thecyclopentadienide ion [C₅H₅(−1)] and various ring substituted and ringfused derivatives, such as the indenylide (−1) ion, that yield a classof bis(cyclopentadieyl)metal compounds, (i.e. the metallocenes); see forexample Robins et al., J. Am. Chem. Soc. 104:1882-1893 (1982); andGassman et al., J. Am. Chem. Soc. 108:4228-4229 (1986), incorporated byreference. Of these, ferrocene [(C₅H₅)₂Fe] and its derivatives areprototypical examples which have been used in a wide variety of chemical(Connelly et al., Chem. Rev. 96:877-910 (1996), incorporated byreference) and electrochemical (Geiger et al., Advances inOrganometallic Chemistry 23:1-93; and Geiger et al., Advances inOrganometallic Chemistry 24:87, incorporated by reference) electrontransfer or “redox” reactions. Metallocene derivatives of a variety ofthe first, second and third row transition metals are potentialcandidates as redox moieties. Other potentially suitable organometallicligands include cyclic arenes such as benzene, to yield bis(arene)metalcompounds and their ring substituted and ring fused derivatives, ofwhich bis(benzene)chromium is a prototypical example, Other acyclicπ-bonded ligands such as the allyl(−1) ion, or butadiene yieldpotentially suitable organometallic compounds, and all such ligands, inconjuction with other π-bonded and δ-bonded ligands constitute thegeneral class of organometallic compounds in which there is a metal tocarbon bond. Electrochemical studies of various dimers and oligomers ofsuch compounds with bridging organic ligands, and additionalnon-bridging ligands, as well as with and without metal-metal bonds arepotential candidate redox moieties.

As described herein, any combination of ligands may be used. Preferredcombinations include: a) all ligands are nitrogen donating ligands; b)all ligands are organometallic ligands; and c) the ligand at theterminus of the conductive oligomer is a metallocene ligand and theligand provided by the binding ligand is a nitrogen donating ligand,with the other ligands, if needed, are either nitrogen donating ligandsor metallocene ligands, or a mixture.

In addition, it may be desirable to use coordination sites of thetransition metal ion for attachment of the redox active molecule toeither a binding ligand (directly or indirectly using a linker), to forma redox active complex, or to the electrode (frequently using a spacersuch as a conductive oligomer, as is more fully described below), orboth. Thus for example, when the redox active molecule is directlyjoined to a binding ligand, one, two or more of the coordination sitesof the metal ion may be occupied by coordination atoms supplied by thebinding ligand (or by the linker, if indirectly joined). In addition, oralternatively, one or more of the coordination sites of the metal ionmay be occupied by a spacer used to attach the redox active molecule tothe electrode.

In addition to transition metal complexes, other organic electron donorsand acceptors may be used in the invention. These organic moleculesinclude, but are not limited to, riboflavin, xanthene dyes, azine dyes,acridine orange, N,N′-dimethyl-2,7-diazapyrenium dichloride (DAP²⁺),methylviologen, ethidium bromide, quinones such asN,N′-dimethylanthra(2,1,9-def:6,5,10-d′e′f′)diisoquinoline dichloride(ADIQ²⁺); porphyrins ([meso-tetrakis(N-methyl-x-pyridinium)porphyrintetrachloride], varlamine blue B hydrochloride, Bindschedler's green;2,6-dichloroindophenol, 2,6-dibromophenolindophenol; Brilliant crestblue (3-amino-9-dimethyl-amino-10-methylphenoxyazine chloride),methylene blue; Nile blue A (aminoaphthodiethylaminophenoxazinesulfate), indigo-5,5′,7,7′-tetrasulfonic acid, indigo-5,5′,7-trisulfonicacid; phenosafranine, indigo-5-monosulfonic acid; safranine T;bis(dimethylglyoximato)-iron(II) chloride; induline scarlet, neutralred, anthracene, coronene, pyrene, 9-phenylanthracene, rubrene,binaphthyl, DPA, phenothiazene, fluoranthene, phenanthrene, chrysene,1,8-diphenyl-1,3,5,7-octatetracene, naphthalene, acenaphthalene,perylene, TMPD and analogs and substituted derivatives of thesecompounds.

In one embodiment, the electron donors and acceptors are redox proteinsas are known in the art. However, redox proteins in many embodiments arenot preferred.

The choice of the specific electron transfer moieties will be influencedby the type of electron transfer detection used, as is generallyoutlined below.

In some embodiments, as is outlined below, the redox active molecule isactually the analyte to be detected; for example, when redox activeproteins such as metalloenzymes, cytochrome c, etc. are to be detected,they may serve as the redox active molecule. Alternatively, some metalanalytes, particularly heavy metals, can also serve as the redox activemolecule, in general with chelating ligands as is described herein; seefor example FIG. 6.

Generally, the target analyte binds to a redox active complex. By “redoxactive complex” herein is meant a complex comprising at least one redoxactive molecule and at least one binding ligand, which, as more fullydescribed below, may be associated in a number of different ways. Insome cases, the binding ligand may also be a redox active molecule. By“binding ligand” or grammatical equivalents herein is meant a compoundthat is used to probe for the presence of the target analyte, and thatwill bind to the analyte.

As will be appreciated by those in the art, the composition of thebinding ligand will depend on the composition of the target analyte.Binding ligands for a wide variety of analytes are known or can bereadily found using known techniques. For example, when the analyte is aprotein, the binding ligands include proteins (particularly includingantibodies or fragments thereof (FAbs, etc.)) or small molecules. Whenthe analyte is a metal ion, the binding ligand generally comprisestraditional metal ion ligands or chelators, which together form theredox active molecule. Preferred binding ligand proteins includepeptides. For example, when the analyte is an enzyme, suitable bindingligands include substrates and inhibitors. Antigen-antibody pairs,receptor-ligands, and carbohydrates and their binding partners are alsosuitable analyte-binding ligand pairs. The binding ligand may be nucleicacid, when nucleic acid binding proteins are the targets; alternatively,as is generally described in U.S. Pat. Nos. 5,270,163, 5,475,096,5,567,588, 5,595,877, 5,637,459, 5,683,867, 5,705,337, and relatedpatents, hereby incorporated by reference, nucleic acid “aptomers” canbe developed for binding to virtually any target analyte. Similarly,there is a wide body of literature relating to the development ofbinding partners based on combinatorial chemistry methods. In thisembodiment, when the binding ligand is a nucleic acid, preferredcompositions and techniques are outlined in PCT US97/20014, herebyincorporated by reference.

By “nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds, although in some cases, as outlined below, nucleic acid analogsare included that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) andreferences therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl etal., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al.,J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437(1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al.,J. Am. Chem. Soc. 111:2321 (1989), O-methylphosphoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al.,Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993);Carlsson et al., Nature 380:207 (1996), all of which are incorporated byreference). Other analog nucleic acids include those with positivebackbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995);non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240,5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed.English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470(1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994);Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modificationsin Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker etal., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) andnon-ribose backbones, including those described in U.S. Pat. Nos.5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580,“Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghuiand P. Dan Cook. Nucleic acids containing one or more carbocyclic sugarsare also included within the definition of nucleic acids (see Jenkins etal., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogsare described in Rawls, C & E News Jun. 2, 1997 page 35. All of thesereferences are hereby expressly incorporated by reference. Thesemodifications of the ribose-phosphate backbone may be done to facilitatethe addition of RAMs or conductive oligomers, or to increase thestability and half-life of such molecules in physiological environments.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made; for example,at the site of conductive oligomer or RAM attachment, an analogstructure may be used. Alternatively, mixtures of different nucleic acidanalogs, and mixtures of naturally occurring nucleic acids and analogsmay be made.

In a preferred embodiment, the binding of the target analyte to thebinding ligand is specific, and the binding ligand is part of a bindingpair. By “specifically bind” herein is meant that the ligand binds theanalyte, with specificity sufficient to differentiate between theanalyte and other components or contaminants of the test sample.However, as will be appreciated by those in the art, it will be possibleto detect analytes using binding which is not highly specific; forexample, the systems may use different binding ligands, for example anarray of different ligands, and detection of any particular analyte isvia its “signature” of binding to a panel of binding ligands, similar tothe manner in which “electronic noses” work. This finds particularutility in the detection of chemical analytes. The binding should besufficient to remain bound under the conditions of the assay, includingwash steps to remove non-specific binding. In some embodiments, forexample in the detection of certain biomolecules, the disassociationconstants of the analyte to the binding ligand will be less than about10⁻⁴-10⁻⁶M⁻¹, with less than about 10⁻⁵ to 10⁻⁹ M⁻¹ being preferred andless than about 10⁻⁷-10⁻⁹ M⁻¹ being particularly preferred.

Together, when present, the redox active molecule and the binding ligandcomprise a redox active complex. As mentioned above, in some cases thebinding ligand is the redox active molecule, and thus the redox activecomplex comprises the redox active binding ligand. Furthermore, in someembodiments, the target analyte is a redox active molecule, such as ametal ion or a metalloenzyme, etc.; in this case, a separate redoxactive molecule need not be used. In addition, there may be more thanone binding ligand or redox active molecule per redox active complex, asis generally outlined below. The redox active complex may also containadditional moieties, such as cross-linking agents, labels, etc., andlinkers for attachment to the electrode. The addition (generally vianon-covalent binding, although as outlined herein, some interactions maybe considered covalent, or post-binding covalent attachment may occur,for example through the use of cross-linking agents) of the targetanalyte to the redox active complex forms an assay complex. By “assaycomplex” herein is meant the complex of components, including targetanalytes, binding ligands and redox active molecules, that allowsdetection. The composition of the assay complex depends on the use ofthe different component outlined herein.

In some embodiments, as is outlined below, the redox active complex issoluble. However, in a preferred embodiment, at least one component ofthe assay complex is covalently attached to an electrode. In a preferredembodiment, it is generally a component of the redox active complex thatis attached; that is, target analytes are not generally covalentlyattached to the electrode. That is, either the redox active molecule orthe binding ligand is covalently attached to the electrode. By“electrode” herein is meant a conductive or semi-conductive composition,which, when connected to an electronic control and detection device, isable to transmit electrons to or from a RAM either in solution or on itssurface. Thus, an electrode is an electron transfer moiety as describedherein. Preferred electrodes are known in the art and include, but arenot limited to, certain metals and their oxides, including gold;platinum; palladium; silicon; aluminum; metal oxide electrodes includingplatinum oxide, titanium oxide, tin oxide, indium tin oxide, palladiumoxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo₂O₆), tungstenoxide (WO₃) and ruthenium oxides; and carbon (including glassy carbonelectrodes, graphite and carbon paste). Preferred electrodes includegold, silicon, carbon and metal oxide electrodes.

The electrodes described herein are depicted as a flat surface, which isonly one of the possible conformations of the electrode and is forschematic purposes only. The conformation of the electrode will varywith the detection method used. For example, flat planar electrodes maybe preferred for optical detection methods, or when arrays of bindingligands are made, thus requiring addressable locations for bothsynthesis and detection. Alternatively, for single probe analysis, theelectrode may be in the form of a tube, with the conductive oligomersand binding ligands bound to the inner surface. This allows a maximum ofsurface area containing the target analytes to be exposed to a smallvolume of sample.

The systems of the invention may take on any number of configurations,as outlined below.

In a preferred embodiment, the system is used to detect pollutants, suchas organic pollutants, as is depicted below in System 1:

In System 1, as is described below, the hatched marks indicate anelectrode, and there is preferably a monolayer on the surface. F₁ is alinkage that allows the covalent attachment of the electrode and theconductive oligomer or insulator, including bonds, atoms or linkers suchas is described herein, for example as “A”, defined below. F₂ is alinkage that allows the covalent attachment of the conductive oligomeror insulator, and may be a bond, an atom or a linkage as is hereindescribed. F₂ may be part of the conductive oligomer, part of theinsulator, part of the terminal group, part of the redox active complexor component, or exogenous to both, for example, as defined herein for“Z”. X is a spacer (conductive oligomer, passivation agent or insulator,as required). RAM is a redox active molecule. TG is a terminal group,which may be chosen to influence the association of the targetpollutant, such as an organic pollutant. Thus for example in thisembodiment TG may be hydrophobic. The association of the pollutant onthe surface will affect the local environment of the RAM, for examplepotentially by changing the E₀ of the RAM or the solvent reorganizationenergy, and thus results in a change in the faradaic impedance of thesystem in the presence of the analyte. The association in this case isnot specific for a particular analyte.

Systems 2, 3, 4 and 5 depict a similar situation except that a specificinteraction is exploited. Thus, the target analyte will bind to thebinding ligand specifically, and is generally large as compared to thebinding ligand and RAM. Upon binding, the local environment of the RAMis affected, for example potentially by changing the E₀ of the RAM orthe solvent reorganization energy, and thus results in a change in thefaradaic impedance of the system in the presence of the analyte. Thetarget analyte in these cases could be protein, a cell, etc. Inaddition, any or all of these systems may be used with co-redoxants, asdescribed below. Upon binding of the target analyte, the access of theco-redoxant to the RAM is restricted, thus resulting in either adifferent signal or a loss in signal, or both. In addition, as for allthe systems depicted herein, the order or proximity of the individualmolecules of the monolayer is not determinative.

In System 2, there may be more than one RAM per binding ligand (BL);that is, the ratio of RAM to BL on the surface (depending on therelative size of the target analyte) may range from 1:1 to over 100:1.This allows an amplification of signal, in that more than one RAM isused to detect a single target analyte.

System 6 depicts a system in which binding of a target analytetheoretically affects the H_(AB) between the RAM and the electrode:

System 7 depicts a similar situation, except that the binding ligand isinherent in the attachment of the RAM to the electrode; for example, itmay be a peptide or nucleic acid to which the analyte binds:

System 8 depicts a situation in which the analyte also serves as theredox active molecule; this is particularly useful in the detection ofmetal ions, for example heavy metal ions, which are toxic. System 8depicts a metal ion, M, and a metal ligand, ML, although as will beappreciated by those in the art, it is quite possible to have theanalyte in this case be a metalloprotein, with a BL, etc. As will beappreciated by those in the art, System 8 is particularly useful in thedetection of different metal ions, using an array of different ligands;preferential binding of one metal over another would result in a panelof results that can be correlated to metal ligand binding. Moreover,different metals may have different E₀s and thus give different signals.

System 9 depicts a competitive-type assay which relies on a decrease insignal for detection. In this case, the target analyte is a ligand, forexample carbon monoxide (CO), which are stronger ligands (SMLs, i.e.have higher binding constants) for a particular metal than the weakermetal ligand (WML) of the system.

System 10 depicts a similar type of assay, which results in a change insignal rather than a decrease in signal. For example, E₀ and λ couldboth change as a result of a new ligand binding.

System 11 utilizes a change in the diffusion coefficient upon analytebinding for the change in faradaic impedance and mass transfer. In thisembodiment, when the ligands are not covalently attached to anelectrode, changes in the diffusion coefficient will alter the masstransfer impedance and thus the total faradaic impedance. That is, insome circumstances the frequency response of a redox active complex willbe limited by its diffusion coefficient. Also, the charge transferimpedance may be altered by the binding of an analyte. At highfrequencies, a redox active complex may not diffuse rapidly enough toreversibly transfer its electron to the electrode at a rate sufficientto generate a strong output signal. At low frequencies, the molecule hassufficient time to diffuse, and thus an output signal can be detected.In this embodiment, the use of monolayers is generally not preferred.

Thus, the result of binding to form an assay complex will generallyalter the diffusion coefficient of the redox active molecule. As aresult, the faradaic impedance will change. This effect will be greatestwhen the binding partner is large in comparison to the redox activemoiety; the redox active moiety will go from being relatively small, andthus diffusing quickly, to relatively large upon

binding into a complex, and diffusing much more slowly; this results inthe greatest changes and is thus preferred. Similarly, binding partnersof roughly equal size can also result in a detectable signal.

Alternatively, it is also possible that binding of the redox activemoiety to its binding partner will cause a decrease in size. Forexample, some protein structures, i.e. antibodies, may have “loose”conformations that are sterically bulky, that “tighten up” as a resultof binding to its partner (i.e. an antigen).

System 11

System 12 is similar to systems 10 and 11, as it is a sensor fordifferent ligands, but it relies on a change in ligands to result in achange in E₀ of the system. A similar system may be used with twometals; that is, instead of adding strong metal ligands, a differentmetal, with different affinity for the ligands may be added, resultingin a electrochemical change.

System 13 is a variation on previous systems, except that the RAM andthe BL are closely associated or linked.

System 14 results in changes in faradaic impedance as a result ofchanges in E₀ or H_(AB). In this case, the binding ligand willself-associate in some way, bringing the RAM into closer proximity tothe electrode. For example, the binding ligand may be a nucleic acid(for example for the detection of a nucleic acid binding protein) or aprotein (for example for the detection of proteins that inhibit or bindthe binding ligand protein. Upon binding of the target, for example aprotein, the conformation and thus the local environment of the RAMchanges, resulting in a detectable signal. System 14 could also be runin “reverse”, wherein the association of the analyte brings the RAM intoproximity of the surface.

System 15 uses two binding ligands, BL1 and BL2, which may be the sameor different, to alter the environment of the RAM. It may be desirableto have one of the binding ligands be a somewhat “generic” bindingligand. Changes in E₀ and/or impedance will result in a detectablesignal.

System 16 also relies on a decrease in signal. In this embodiment, atarget analyte is used that will bind the metal ion-binding ligandcomplex in such a way as to render the metal unavailable to serve as aredox active molecule.

System 17 utilizes a change in metal ion affinity to a particularbinding ligand to detect a change in the signal based on a differentmetal being present (resulting in a different E_(o)).

System 18 is similar to System 9 and depicts a competitive-type assayfor detecting a target analyte. In System 15, a covalently attachedtarget analyte or target analog (TA) is competed off of the bindingligand by the addition of the target analyte, resulting in a decrease insignal.

System 19 is a mixture of Systems 2 and 18, where the replacement of abulky analog (TA) by a smaller target analyte (T) results in a differentsignal. For example, co-redoxant reactions could now occur.Alternatively, monolayers with “holes”, that would allow current flow inthe absence of the analog but do not in its presence, could also beused.

System 20 depicts a two electrode system in a competitive-type assay.This is useful in that it allows detection of an increase in signal onthe second electrode, which is generally preferable to the loss of asignal.

As will be appreciated by those in the art, System 20 may also beconfigured in several different ways. BL1 and BL2 may have differentaffinities for the same site on the target analyte or analog, or bind todifferent sites. Similarly, the other systems may also be run in twoelectrode systems.

In addition, it is possible to use systems like those depicted above inseveral other embodiments. For example, since heat will change thefaradaic impedance, the systems above could be used as a heat sensor.Similarly, attachment of the RAM to the electrode using a labile orcleavable bond can allow sensing of the cleaving agent based on adecrease in signal; for example, photolabile bonds can be used to detectlight (uv); substrates can be used to sense enzymes (proteases,nucleases, carbohydrases, lipases, etc.) or other cleaving agents, suchas drugs that cut nucleic acids, etc.

In the systems described above, the redox active complex is covalentlyattached to the electrode. This may be accomplished in any number ofways, as will be apparent to those in the art. In a preferredembodiment, one or both of the redox active molecule and the bindingligand are attached, via a spacer, to the electrode, using thetechniques and compositions outlined below. By “spacer” herein is meanta moiety which holds the redox active complex off the surface of theelectrode. In a preferred embodiment, the spacer used to attach theredox active molecule is a conductive oligomer as outlined herein,although suitable spacer moieties include passivation agents andinsulators as outlined below. The spacer moieties may be substantiallynon-conductive. In general, the length of the spacer is as outlined forconductive polymers and passivation agents. As will be appreciated bythose in the art, if the spacer becomes too long, the electroniccoupling between the redox active molecule and the electrode willdecrease rapidly.

In a preferred embodiment, the redox active molecule will be attachedvia a conductive oligomer, such that detection of changes in faradaicimpedance as between the redox active molecule and the electrode can bedetected. Other components of the system may be attached using otherspacers; for example, when the binding ligand and the redox activemolecule are attached separately, as is generally depicted in System 2,the binding ligand may be attached via a non-conductive oligomer spacer.

In a preferred embodiment, the spacer is a conductive oligomer. By“conductive oligomer” herein is meant a substantially conductingoligomer, preferably linear, some embodiments of which are referred toin the literature as “molecular wires”.

By “conductive oligomer” herein is meant a substantially conductingoligomer, preferably linear, some embodiments of which are referred toin the literature as “molecular wires”. By “substantially conducting”herein is meant that the rate of electron transfer through theconductive oligomer is generally not the rate limiting step in thedetection of the target analyte, although as noted below, systems whichuse spacers that are the rate limiting step are also acceptable. Stateddifferently, the resistance of the conductive oligomer is less than thatof the other components of the system. Generally, the conductiveoligomer has substantially overlapping π-orbitals, i.e. conjugatedπ-orbitals, as between the monomeric units of the conductive oligomer,although the conductive oligomer may also contain one or more sigma (a)bonds. Additionally, a conductive oligomer may be defined functionallyby its ability to pass electrons into or from an attached component.Furthermore, the conductive oligomer is more conductive than theinsulators as defined herein.

In a preferred embodiment, the conductive oligomers have a conductivity,S, of from between about 10⁻⁶ to about 10^(4 Ω) ⁻¹cm⁻¹, with from about10⁻⁵ to about 10^(3 Ω) ⁻¹cm⁻¹ being preferred, with these S values beingcalculated for molecules ranging from about 20 {acute over (Å)} to about200 {acute over (Å)}. As described below, insulators have a conductivityS of about 10^(−7 Ω) ⁻¹ cm⁻¹ or lower, with less than about 10^(−8 Ω)⁻¹cm⁻¹ being preferred. See generally Gardner et al., Sensors andActuators A 51 (1995) 57-66, incorporated herein by reference.

Desired characteristics of a conductive oligomer include highconductivity, sufficient solubility in organic solvents and/or water forsynthesis and use of the compositions of the invention, and preferablychemical resistance to reactions that occur i) during synthesis of thesystems of the invention, ii) during the attachment of the conductiveoligomer to an electrode, or iii) during test assays.

The oligomers of the invention comprise at least two monomeric subunits,as described herein. As is described more fully below, oligomers includehomo- and hetero-oligomers, and include polymers.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 1:

As will be understood by those in the art, all of the structuresdepicted herein may have additional atoms or structures; i.e. theconductive oligomer of Structure 1 may be attached to redox activemolecules such as electrodes, transition metal complexes, organicelectron transfer moieties, and metallocenes, and to binding ligands, orto several of these. Unless otherwise noted, the conductive oligomersdepicted herein will be attached at the left side to an electrode; thatis, as depicted in Structure 1, the left “Y” is connected to theelectrode as described herein and the right “Y”, if present, is attachedto the redox active complex, either directly or through the use of alinker, as is described herein.

In this embodiment, Y is an aromatic group, n is an integer from 1 to50, g is either 1 or zero, e is an integer from zero to 10, and m iszero or 1. When g is 1, B-D is a conjugated bond, preferably selectedfrom acetylene, alkene, substituted alkene, amide, azo, —C═N-(including—N═C—, —CR═N— and —N═CR—), —Si═Si—, and —Si═C— (including —C═Si—,—Si═CR— and —CR═Si—). When g is zero, e is preferably 1, D is preferablycarbonyl, or a heteroatom moiety, wherein the heteroatom is selectedfrom oxygen, sulfur, nitrogen or phosphorus. Thus, suitable heteroatommoieties include, but are not limited to, —NH and —NR, wherein R is asdefined herein; substituted sulfur; sulfonyl (—SO₂—) sulfoxide (—SO—);phosphine oxide (—PO— and —RPO—); and thiophosphine (—PS— and —RPS—).However, when the conductive oligomer is to be attached to a goldelectrode, as outlined below, sulfur derivatives are not preferred.

By “aromatic group” or grammatical equivalents herein is meant anaromatic monocyclic or polycyclic hydrocarbon moiety generallycontaining 5 to 14 carbon atoms (although larger polycyclic ringsstructures may be made) and any carbocylic ketone or thioketonederivative thereof, wherein the carbon atom with the free valence is amember of an aromatic ring. Aromatic groups include arylene groups andaromatic groups with more than two atoms removed. For the purposes ofthis application aromatic includes heterocycle. “Heterocycle” or“heteroaryl” means an aromatic group wherein 1 to 5 of the indicatedcarbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen,sulfur, phosphorus, boron and silicon wherein the atom with the freevalence is a member of an aromatic ring, and any heterocyclic ketone andthioketone derivative thereof. Thus, heterocycle includes thienyl,furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl,isoquinolyl, thiazolyl, imidozyl, etc.

Importantly, the Y aromatic groups of the conductive oligomer may bedifferent, i.e. the conductive oligomer may be a heterooligomer. Thatis, a conductive oligomer may comprise a oligomer of a single type of Ygroups, or of multiple types of Y groups. Thus, in a preferredembodiment, when a barrier monolayer is used as is described below, oneor more types of Y groups are used in the conductive oligomer within themonolayer with a second type(s) of Y group used above the monolayerlevel. Thus, as is described herein, the conductive oligomer maycomprise Y groups that have good packing efficiency within the monolayerat the electrode surface, and a second type(s) of Y groups with greaterflexibility and hydrophilicity above the monolayer level to facilitatetarget analyte binding. For example, unsubstituted benzyl rings maycomprise the Y rings for monolayer packing, and substituted benzyl ringsmay be used above the monolayer. Alternatively, heterocylic rings,either substituted or unsubstituted, may be used above the monolayer.Additionally, in one embodiment, heterooligomers are used even when theconductive oligomer does not extend out of the monolayer.

The aromatic group may be substituted with a substitution group,generally depicted herein as R. R groups may be added as necessary toaffect the packing of the conductive oligomers, i.e. when the conductiveoligomers form a monolayer on the electrode, R groups may be used toalter the association of the oligomers in the monolayer. R groups mayalso be added to 1) alter the solubility of the oligomer or ofcompositions containing the oligomers; 2) alter the conjugation orelectrochemical potential of the system; and 3) alter the charge orcharacteristics at the surface of the monolayer.

In a preferred embodiment, when the conductive oligomer is greater thanthree subunits, R groups are preferred to increase solubility whensolution synthesis is done. However, the R groups, and their positions,are chosen to minimally effect the packing of the conductive oligomerson a surface, particularly within a monolayer, as described below. Ingeneral, only small R groups are used within the monolayer, with largerR groups generally above the surface of the monolayer. Thus for examplethe attachment of methyl groups to the portion of the conductiveoligomer within the monolayer to increase solubility is preferred, withattachment of longer alkoxy groups, for example, C3 to C10, ispreferably done above the monolayer surface. In general, for the systemsdescribed herein, this generally means that attachment of stericallysignificant R groups is not done on any of the first three oligomersubunits, depending on the length of the insulator molecules.

Suitable R groups include, but are not limited to, hydrogen, alkyl,alcohol, aromatic, amino, amido, nitro, ethers, esters, aldehydes,sulfonyl, silicon moieties, halogens, sulfur containing moieties,phosphorus containing moieties, and ethylene glycols. In the structuresdepicted herein, R is hydrogen when the position is unsubstituted. Itshould be noted that some positions may allow two substitution groups, Rand R′, in which case the R and R′ groups may be either the same ordifferent.

By “alkyl group” or grammatical equivalents herein is meant a straightor branched chain alkyl group, with straight chain alkyl groups beingpreferred. If branched, it may be branched at one or more positions, andunless specified, at any position. The alkyl group may range from about1 to about 30 carbon atoms (C1-C30), with a preferred embodimentutilizing from about 1 to about 20 carbon atoms (C1-C20), with about C1through about C12 to about C15 being preferred, and C1 to C5 beingparticularly preferred, although in some embodiments the alkyl group maybe much larger. Also included within the definition of an alkyl groupare cycloalkyl groups such as C5 and C6 rings, and heterocyclic ringswith nitrogen, oxygen, sulfur or phosphorus. Alkyl also includesheteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and siliconebeing preferred. Alkyl includes substituted alkyl groups. By“substituted alkyl group” herein is meant an alkyl group furthercomprising one or more substitution moieties “R”, as defined above.

By “amino groups” or grammatical equivalents herein is meant —NH₂, —NHRand —NR₂ groups, with R being as defined herein.

By “nitro group” herein is meant an —NO₂ group.

By “sulfur containing moieties” herein is meant compounds containingsulfur atoms, including but not limited to, thia-, thio- andsulfo-compounds, thiols (—SH and —SR), and sulfides (—RSR—). By“phosphorus containing moieties” herein is meant compounds containingphosphorus, including, but not limited to, phosphines and phosphates. By“silicon containing moieties” herein is meant compounds containingsilicon.

By “ether” herein is meant an —O—R group. Preferred ethers includealkoxy groups, with —O—(CH₂)₂CH₃ and —O—(CH₂)₄CH₃ being preferred.

By “ester” herein is meant a —COOR group.

By “halogen” herein is meant bromine, iodine, chlorine, or fluorine.Preferred substituted alkyls are partially or fully halogenated alkylssuch as CF₃, etc.

By “aldehyde” herein is meant —RCOH groups.

By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH.

By “amido” herein is meant —RCONH— or RCONR— groups.

By “ethylene glycol” herein is meant a —(O—CH₂—CH₂)_(n)— group, althougheach carbon atom of the ethylene group may also be singly or doublysubstituted, i.e. —(O—CR₂—CR₂)_(n), with R as described above. Ethyleneglycol derivatives with other heteroatoms in place of oxygen (i.e.—(N—CH₂—CH₂)_(n)— or —(S—CH₂—CH₂)_(n)—, or with substitution groups) arealso preferred.

Preferred substitution groups include, but are not limited to, methyl,ethyl, propyl, alkoxy groups such as —O—(CH₂)₂CH₃ and —O—(CH₂)₄CH₃ andethylene glycol and derivatives thereof.

Preferred aromatic groups include, but are not limited to, phenyl,naphthyl, naphthalene, anthracene, phenanthroline, pyrole, pyridine,thiophene, porphyrins, and substituted derivatives of each of these,included fused ring derivatives.

In the conductive oligomers depicted herein, when g is 1, B-D is a bondlinking two atoms or chemical moieties. In a preferred embodiment, B-Dis a conjugated bond, containing overlapping or conjugated π-orbitals.

Preferred B-D bonds are selected from acetylene (—C≡C—, also calledalkyne or ethyne), alkene (—CH═CH—, also called ethylene), substitutedalkene (—CR═CR—, —CH═CR— and —CR═CH—), amide (—NH—CO— and —NR—CO— or—CO—NH— and —CO—NR—), azo (—N═N—), esters and thioesters (—CO—O—,—O—CO—, —CS—O— and —O—CS—) and other conjugated bonds such as (—CH═N—,—CR═N—, —N═CH— and —N═CR—), (—SiH═SiH—, —SiR═SiH—, —SiR═SiH—, and—SiR═SiR—), (—SiH═CH—, —SiR═CH—, —SiH═CR—, —SiR═CR—, —CH═SiH—, —CR═SiH—,—CH═SiR—, and —CR═SiR—). Particularly preferred B-D bonds are acetylene,alkene, amide, and substituted derivatives of these three, and azo.Especially preferred B-D bonds are acetylene, alkene and amide. Theoligomer components attached to double bonds may be in the trans or cisconformation, or mixtures. Thus, either B or D may include carbon,nitrogen or silicon. The substitution groups are as defined as above forR.

When g=0 in the Structure 2 conductive oligomer, e is preferably 1 andthe D moiety may be carbonyl or a heteroatom moiety as defined above.

As above for the Y rings, within any single conductive oligomer, the B-Dbonds (or D moieties, when g=0) may be all the same, or at least one maybe different. For example, when m is zero, the terminal B-D bond may bean amide bond, and the rest of the B-D bonds may be acetylene bonds.Generally, when amide bonds are present, as few amide bonds as possibleare preferable, but in some embodiments all the B-D bonds are amidebonds. Thus, as outlined above for the Y rings, one type of B-D bond maybe present in the conductive oligomer within a monolayer as describedbelow, and another type above the monolayer level, to give greaterflexibility.

In the structures depicted herein, n is an integer from 1 to 50,although longer oligomers may also be used (see for example Schumm etal., Angew. Chem. Int. Ed. Engl. 1994 33(13):1360). Without being boundby theory, it appears that for efficient binding of target analytes on asurface, the binding should occur at a distance from the surface, i.e.the kinetics of binding increase as a function of the distance from thesurface, particularly for larger analytes. Accordingly, the length ofthe conductive oligomer is such that the closest portion of the redoxactive complex is positioned from about 6 {acute over (Å)} to about 100{acute over (Å)} (although distances of up to 500 {acute over (Å)} maybe used) from the electrode surface, with from about 25 {acute over (Å)}to about 60 {acute over (Å)} being preferred. In a preferred embodiment,the length of the conductive oligomer is greater than (CH₂)₆ linkers,with greater than (CH₂)₁₀ being preferred and greater than about (CH₂)₁₆being particularly preferred. Accordingly, n will depend on the size ofthe aromatic group, but generally will be from about 1 to about 20, withfrom about 2 to about 15 being preferred and from about 3 to about 10being especially preferred.

In the structures depicted herein, m is either 0 or 1. That is, when mis 0, the conductive oligomer may terminate in the B-D bond or D moiety,i.e. the D atom is attached to the component of the redox active complexeither directly or via a linker. In some embodiments, there may beadditional atoms, such as a linker, attached between the conductiveoligomer and the component of the redox active complex to which it isattached. Additionally, as outlined below, the D atom may be a nitrogenatom of a redox active complex, for example an amine of a protein.Alternatively, when m is 1, the conductive oligomer may terminate in Y,an aromatic group, i.e. the aromatic group is attached to the redoxactive complex or linker.

As will be appreciated by those in the art, a large number of possibleconductive oligomers may be utilized. These include conductive oligomersfalling within the Structure 1 and Structure 8 formulas, as well asother conductive oligomers, as are generally known in the art, includingfor example, compounds comprising fused aromatic rings or Teflon®-likeoligomers, such as —(CF₂)_(n)—, —(CHF)_(n)— and —(CFR)_(n)—. See forexample, Schumm et al., angew. Chem. Intl. Ed. Engl. 33:1361 (1994);Grosshenny et al., Platinum Metals Rev. 40(1):26-35 (1996); Tour, Chem.Rev. 96:537-553 (1996); Hsung et al., Organometallics 14:4808-4815(1995; and references cited therein, all of which are expresslyincorporated by reference.

Particularly preferred conductive oligomers of this embodiment aredepicted below:

Structure 2 is Structure 1 when g is 1. Preferred embodiments ofStructure 2 include: e is zero, Y is pyrole or substituted pyrole; e iszero, Y is thiophene or substituted thiophene; e is zero, Y is furan orsubstituted furan; e is zero, Y is phenyl or substituted phenyl; e iszero, Y is pyridine or substituted pyridine; e is 1, B-D is acetyleneand Y is phenyl or substituted phenyl (see Structure 4 below). Apreferred embodiment of Structure 2 is also when e is one, depicted asStructure 3 below:

Preferred embodiments of Structure 3 are: Y is phenyl or substitutedphenyl and B-D is azo; Y is phenyl or substituted phenyl and B-D isalkene; Y is pyridine or substituted pyridine and B-D is acetylene; Y isthiophene or substituted thiophene and B-D is acetylene; Y is furan orsubstituted furan and B-D is acetylene; Y is thiophene or furan (orsubstituted thiophene or furan) and B-D are alternating alkene andacetylene bonds.

Most of the structures depicted herein utilize a Structure 3 conductiveoligomer. However, any Structure 4 oligomers may be substituted with aStructure 1, 2 or 8 oligomer, or other conducting oligomer, and the useof such Structure 3 depiction is not meant to limit the scope of theinvention.

Particularly preferred embodiments of Structure 3 include Structures 4,5, 6 and 7, depicted below:

Particularly preferred embodiments of Structure 4 include: n is two, mis one, and R is hydrogen; n is three, m is zero, and R is hydrogen; andthe use of R groups to increase solubility.

When the B-D bond is an amide bond, as in Structure 5, the conductiveoligomers are pseudopeptide oligomers. Although the amide bond inStructure 5 is depicted with the carbonyl to the left, i.e. —CONH—, thereverse may also be used, i.e. —NHCO—. Particularly preferredembodiments of Structure 5 include: n is two, m is one, and R ishydrogen; n is three, m is zero, and R is hydrogen (in this embodiment,the terminal nitrogen (the D atom) may be the nitrogen of theamino-modified ribose); and the use of R groups to increase solubility.

Preferred embodiments of Structure 6 include the first n is two, secondn is one, m is zero, and all R groups are hydrogen, or the use of Rgroups to increase solubility.

Preferred embodiments of Structure 7 include: the first n is three, thesecond n is from 1-3, with m being either 0 or 1, and the use of Rgroups to increase solubility.

In a preferred embodiment, the conductive oligomer has the structuredepicted in Structure 8:

In this embodiment, C are carbon atoms, n is an integer from 1 to 50, mis 0 or 1, J is a heteroatom selected from the group consisting ofnitrogen, silicon, phosphorus, sulfur, carbonyl or sulfoxide, and G is abond selected from alkane, alkene or acetylene, such that together withthe two carbon atoms the C-G-C group is an alkene (—CH═CH—), substitutedalkene (—CR═CR—) or mixtures thereof (—CH═CR— or —CR═CH—), acetylene(—C≡C—), or alkane (—CR₂—CR₂—, with R being either hydrogen or asubstitution group as described herein). The G bond of each subunit maybe the same or different than the G bonds of other subunits; that is,alternating oligomers of alkene and acetylene bonds could be used, etc.However, when G is an alkane bond, the number of alkane bonds in theoligomer should be kept to a minimum, with about six or less sigma bondsper conductive oligomer being preferred. Alkene bonds are preferred, andare generally depicted herein, although alkane and acetylene bonds maybe substituted in any structure or embodiment described herein as willbe appreciated by those in the art.

In a preferred embodiment, the m of Structure 8 is zero. In aparticularly preferred embodiment, m is zero and G is an alkene bond, asis depicted in Structure 9:

The alkene oligomer of structure 9, and others depicted herein, aregenerally depicted in the preferred trans configuration, althougholigomers of cis or mixtures of trans and cis may also be used. Asabove, R groups may be added to alter the packing of the compositions onan electrode, the hydrophilicity or hydrophobicity of the oligomer, andthe flexibility, i.e. the rotational, torsional or longitudinalflexibility of the oligomer. n is as defined above.

In a preferred embodiment, R is hydrogen, although R may be also alkylgroups and polyethylene glycols or derivatives.

In an alternative embodiment, the conductive oligomer may be a mixtureof different types of oligomers, for example of structures 1 and 8.

The conductive oligomers are covalently attached to a component of theredox active complex, either the binding ligand, or the redox activemolecule, or both, as is generally outlined in the systems describedabove. By “covalently attached” herein is meant that two moieties areattached by at least one bond, including sigma bonds, pi bonds andcoordination bonds.

The method of attachment of the redox active complex to the spacer (alsosometimes referred to herein as an attachment linker, which may beeither an insulator or conductive oligomer) will generally be done as isknown in the art, and will depend on both the composition of theattachment linker and the capture binding ligand. In general, the redoxactive complexes are attached to the attachment linker through the useof functional groups on each that can then be used for attachment.Preferred functional groups for attachment are amino groups, carboxygroups, oxo groups and thiol groups. These functional groups can then beattached, either directly or indirectly through the use of a linker,sometimes depicted herein as “Z”. Linkers are well known in the art; forexample, homo- or hetero-bifunctional linkers as are well known (see1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference).Preferred Z linkers include, but are not limited to, alkyl groups(including substituted alkyl groups and alkyl groups containingheteroatom moieties), with short alkyl groups, esters, amide, amine,epoxy groups and ethylene glycol and derivatives being preferred, withpropyl, acetylene, and C₂ alkene being especially preferred. Z may alsobe a sulfone group, forming sulfonamide linkages.

A preferred attachment of redox active molecules that are transitionmetal complexes utilizes either a transition metal ligand (includingcoordination atoms) on the terminus of the conductive oligomer, thatserves to attach the redox active molecule to the conductive oligomer,as is generally depicted below in Structures 10 and 11. Both Structure10 and 11 depict a structure 3 conductive oligomer, although otheroligomers may be used. Similarly, if a binding ligand is attached (forexample as shown in System 4), the metal ligand can either be attachedto the binding ligand (i.e. exogeneously added, for example using a Zlinker) or can be contributed by the binding ligand itself (for example,using a nitrogen of an amino acid side chain).

L are the co-ligands, that provide the coordination atoms for thebinding of the metal ion. As will be appreciated by those in the art,the number and nature of the co-ligands will depend on the coordinationnumber of the metal ion. Mono-, di- or polydentate co-ligands may beused at any position. Thus, for example, when the metal has acoordination number of six, the L from the terminus of the conductiveoligomer, the L contributed from the binding ligand (if present) and r,add up to six. Thus, when the metal has a coordination number of six, rmay range from zero (when all coordination atoms are provided by theother ligands) to five, when all the co-ligands are monodentate. Thusgenerally, r will be from 0 to 8, depending on the coordination numberof the metal ion and the choice of the other ligands.

In one embodiment, the metal ion has a coordination number of six andboth the ligand attached to the conductive oligomer and the ligandeither attached or contributed by the binding ligand are at leastbidentate; that is, r is preferably zero, one (i.e. the remainingco-ligand is bidentate) or two (two monodentate co-ligands are used).

As will be appreciated in the art, the co-ligands can be the same ordifferent.

When one or more of the co-ligands is an organometallic ligand, theligand is generally attached via one of the carbon atoms of theorganometallic ligand, although attachment may be via other atoms forheterocyclic ligands. Preferred organometallic ligands includemetallocene ligands, including substituted derivatives and themetalloceneophanes (see page 1174 of Cotton and Wilkenson, supra). Forexample, derivatives of metallocene ligands such asmethylcyclopentadienyl, with multiple methyl groups being preferred,such as pentamethylcyclopentadienyl, can be used to increase thestability of the metallocene. In a preferred embodiment, only one of thetwo metallocene ligands of a metallocene are derivatized.

In this way, redox active complexes may be attached, including bindingligands comprising proteins, lectins, nucleic acids, small organicmolecules, carbohydrates, etc.

A preferred embodiment utilizes proteinaceous binding ligands. As isknown in the art, any number of techniques may be used to attach aproteinaceous binding ligand to an attachment linker, as is outlinedabove for the attachment of a redox active complex to the spacer; seealso FIGS. 2 and 3. A wide variety of techniques are known to addmoieties to proteins. Similar techniques can be used to add the bindingligand to the redox active molecule, for example as depicted in System3, 4 or 5, as will be appreciated by those in the art.

A preferred embodiment utilizes nucleic acids as the binding ligand,with techniques outlined in PCT US97/20014 being useful for attachment.

One end of the attachment linker is linked to the redox active complex,and the other end (although as will be appreciated by those in the art,it need not be the exact terminus for either) is attached to theelectrode. Thus, any of structures depicted herein may further comprisea redox active complex or system component effectively as a terminalgroup.

The covalent attachment of the conductive oligomer containing the redoxactive molecule (and the attachment of other spacer molecules) may beaccomplished in a variety of ways, depending on the electrode and theconductive oligomer used. Generally, some type of linker is used, asdepicted below as “A” in Structure 12, where X is the conductiveoligomer, and the hatched surface is the electrode:

In this embodiment, A is a linker or atom. The choice of “A” will dependin part on the characteristics of the electrode. Thus, for example, Amay be a sulfur moiety when a gold electrode is used. Alternatively,when metal oxide electrodes are used, A may be a silicon (silane) moietyattached to the oxygen of the oxide (see for example Chen et al.,Langmuir 10:3332-3337 (1994); Lenhard et al., J. Electroanal. Chem.78:195-201 (1977), both of which are expressly incorporated byreference). When carbon based electrodes are used, A may be an aminomoiety (preferably a primary amine; see for example Deinhammer et al.,Langmuir 10:1306-1313 (1994)). Thus, preferred A moieties include, butare not limited to, silane moieties, sulfur moieties (including alkylsulfur moieties), and amino moieties. In a preferred embodiment, epoxidetype linkages with redox polymers such as are known in the art are notused.

Although depicted herein as a single moiety, the conductive oligomer(and other spacers) may be attached to the electrode with more than one“A” moiety; the “A” moieties may be the same or different. Thus, forexample, when the electrode is a gold electrode, and “A” is a sulfuratom or moiety, such as generally depicted below in Structure 13,multiple sulfur atoms may be used to attach the conductive oligomer tothe electrode, such as is generally depicted below in Structures 14, 15and 16. As will be appreciated by those in the art, other suchstructures can be made. In Structures 14, 15 and 16, the A moiety isjust a sulfur atom, but substituted sulfur moieties may also be used.

It should also be noted that similar to Structure 16, it may be possibleto have a conductive oligomer terminating in a single carbon atom withthree sulfur moities attached to the electrode.

In a preferred embodiment, the electrode is a gold electrode, andattachment is via a sulfur linkage as is well known in the art, i.e. theA moiety is a sulfur atom or moiety. Although the exact characteristicsof the gold-sulfur attachment are not known, this linkage is consideredcovalent for the purposes of this invention. A representative structureis depicted in Structure 17. Structure 17 depicts the “A” linker ascomprising just a sulfur atom, although additional atoms may be present(i.e. linkers from the sulfur to the conductive oligomer or substitutiongroups).

In a preferred embodiment, the electrode is a carbon electrode, i.e. aglassy carbon electrode, and attachment is via a nitrogen of an aminegroup. A representative structure is depicted in Structure 18. Again,additional atoms may be present, i.e. Z type linkers.

In Structure 19, the oxygen atom is from the oxide of the metal oxideelectrode. The Si atom may also contain other atoms, i.e. be a siliconmoiety containing substitution groups.

Thus, in a preferred embodiment, electrodes are made that compriseconductive oligomers attached to redox active complexes for the purposesof hybridization assays, as is more fully described herein. As will beappreciated by those in the art, electrodes can be made that have asingle species of binding ligand, i.e. for the detection of a singletarget analyte, or multiple binding ligand species, i.e. for thedetection of multiple target analytes.

In addition, as outlined herein, the use of a solid support such as anelectrode enables the use of these assays in an array form. The use ofarrays such as oligonucleotide arrays are well known in the art, andsimilar systems can be built herein. In addition, techniques are knownfor “addressing” locations within an electrode array and for the surfacemodification of electrodes. Thus, in a preferred embodiment, arrays ofdifferent binding ligands are laid down on the array of electrodes, eachof which are covalently attached to the electrode via a conductivelinker. In this embodiment, the number of different species of analytesmay vary widely, from one to thousands, with from about 4 to about100,000 being preferred, and from about 10 to about 10,000 beingparticularly preferred.

In a preferred embodiment, the electrode further comprises a passayationagent, preferably in the form of a monolayer on the electrode surface.The efficiency of binding may increase when the target analyte is at adistance from the electrode, and non-specific binding is decreased whena monolayer is used. A passayation agent layer facilitates themaintenance of the target analyte away from the electrode surface. Inaddition, a passayation agent serves to keep charge carriers away fromthe surface of the electrode. Thus, this layer helps to preventelectrical contact between the electrodes and the electron transfermoieties, or between the electrode and charged species within thesolvent. Such contact can result in a direct “short circuit” or anindirect short circuit via charged species which may be present in thesample. Accordingly, the monolayer of passayation agents is preferablytightly packed in a uniform layer on the electrode surface, such that aminimum of “holes” exist. Alternatively, the passayation agent may notbe in the form of a monolayer, but may be present to help the packing ofthe conductive oligomers or other characteristics.

The passayation agents thus serve as a physical barrier to block solventaccessibility to the electrode. As such, the passayation agentsthemselves may in fact be either (1) conducting or (2) nonconducting,i.e. insulating, molecules. Thus, in one embodiment, the passayationagents are conductive oligomers, as described herein, with or without aterminal group to block or decrease the transfer of charge to theelectrode. Other passayation agents include oligomers of —(CF₂)_(n)—,—(CHF)_(n)— and —(CFR)_(n)—. In a preferred embodiment, the passayationagents are insulator moieties.

An “insulator” is a substantially nonconducting oligomer, preferablylinear. By “substantially nonconducting” herein is meant that the rateof electron transfer through the insulator is the rate limiting step ofthe transfer reaction. Stated differently, the electrical resistance ofthe insulator is higher than the electrical resistance of the rest ofthe system. The rate of electron transfer through the insulator ispreferably slower than the rate through the conductive oligomersdescribed herein. It should be noted however that even oligomersgenerally considered to be insulators still may transfer electrons,albeit at a slow rate.

In a preferred embodiment, the insulators have a conductivity, S, ofabout 10^(−7 Ω) ⁻¹cm⁻¹ or lower, with less than about 10^(−8 Ω) ⁻¹cm⁻¹being preferred. See generally Gardner et al., supra.

Generally, insulators are alkyl or heteroalkyl oligomers or moietieswith sigma bonds, although any particular insulator molecule may containaromatic groups or one or more conjugated bonds. By “heteroalkyl” hereinis meant an alkyl group that has at least one heteroatom, i.e. nitrogen,oxygen, sulfur, phosphorus, silicon or boron included in the chain.Alternatively, the insulator may be quite similar to a conductiveoligomer with the addition of one or more heteroatoms or bonds thatserve to inhibit or slow, preferably substantially, electron transfer.

The passayation agents, including insulators, may be substituted with Rgroups as defined herein to alter the packing of the moieties orconductive oligomers on an electrode, the hydrophilicity orhydrophobicity of the insulator, and the flexibility, i.e. therotational, torsional or longitudinal flexibility of the insulator. Forexample, branched alkyl groups may be used. In addition, the terminus ofthe passayation agent, including insulators, may contain an additionalgroup to influence the exposed surface of the monolayer. For example,there may be negatively charged groups on the terminus to form anegatively charged surface to repel negatively charged species fromnon-specifically binding. Similarly, for example as depicted in System1, hydrophobic groups can be used to attract hydrophobic analytes, etc.Preferred passayation agent terminal groups include —NH₂, —OH, —COOH,—CH₃, trimethylsilyl (TMS) and (poly)ethylene glycol, with the latterbeing particularly preferred.

The length of the passayation agent will vary as needed. As outlinedabove, it appears that binding is more efficient at a distance from thesurface. Thus, the length of the passayation agents is similar to thelength of the conductive oligomers, as outlined above. In addition, theconductive oligomers may be basically the same length as the passayationagents or longer than them, resulting in the binding ligands being moreaccessible to the solvent for binding of target analytes.

The monolayer may comprise a single type of passayation agent, includinginsulators, or different types.

Suitable insulators are known in the art, and include, but are notlimited to, —(CH₂)_(n)—, —(CRH)_(n)—, and —(CR₂)_(n)—, ethylene glycolor derivatives using other heteroatoms in place of oxygen, i.e. nitrogenor sulfur (sulfur derivatives are not preferred when the electrode isgold).

The passayation agents are generally attached to the electrode in thesame manner as the conductive oligomer, and may use the same “A” linkeras defined above.

The compositions of the invention are generally synthesized as outlinedbelow, generally utilizing techniques well known in the art.

The compositions may be made in several ways. A preferred method firstsynthesizes a conductive oligomer attached to the binding ligand orredox active molecule, followed by attachment to the electrode. Thesecond component of the redox active complex may be added prior toattachment to the electrode or after. Alternatively, the redox activecomplex may be made and then the completed conductive oligomer added,followed by attachment to the electrode. Alternatively, the conductiveoligomer and monolayer (if present) are attached to the electrode first,followed by attachment of the other components.

In a preferred embodiment, conductive oligomers are covalently attachedvia sulfur linkages to the electrode. However, surprisingly, traditionalprotecting groups for use of attaching molecules to gold electrodes aregenerally not ideal for use in both synthesis of the compositionsdescribed herein and inclusion in biomolecule synthetic reactions.Accordingly, alternate methods for the attachment of conductiveoligomers to gold electrodes, utilizing unusual protecting groups,including ethylpyridine, and trimethylsilylethyl as is described in PCTUS97/20014. Briefly, in a preferred embodiment, the subunit of theconductive oligomer which contains the sulfur atom for attachment to theelectrode is protected with an ethyl-pyridine or trimethylsilylethylgroup. For the former, this is generally done by contacting the subunitcontaining the sulfur atom (preferably in the form of a sulfhydryl) witha vinyl pyridine group or vinyl trimethylsilylethyl group underconditions whereby an ethylpyridine group or trimethylsilylethyl groupis added to the sulfur atom.

This subunit also generally contains a functional moiety for attachmentof additional subunits, and thus additional subunits are attached toform the conductive oligomer. The conductive oligomer is then attachedto a component of the redox active complex. The protecting group is thenremoved and the sulfur-gold covalent attachment is made. Alternatively,all or part of the conductive oligomer is made, and then either asubunit containing a protected sulfur atom is added, or a sulfur atom isadded and then protected. The conductive oligomer is then attached to acomponent of the redox active complex. Alternatively, the conductiveoligomer attached to the redox active complex component, and then eithera subunit containing a protected sulfur atom is added, or a sulfur atomis added and then protected. Alternatively, the ethyl pyridineprotecting group may be used as above, but removed after one or moresteps and replaced with a standard protecting group like a disulfide.Thus, the ethyl pyridine or trimethylsilylethyl group may serve as theprotecting group for some of the synthetic reactions, and then removedand replaced with a traditional protecting group.

By “subunit” of a conductive polymer herein is meant at least the moietyof the conductive oligomer to which the sulfur atom is attached,although additional atoms may be present, including either functionalgroups which allow the addition of additional components of theconductive oligomer, or additional components of the conductiveoligomer. Thus, for example, when Structure 1 oligomers are used, asubunit comprises at least the first Y group.

A preferred method comprises 1) adding an ethyl pyridine ortrimethylsilylethyl protecting group to a sulfur atom attached to afirst subunit of a conductive oligomer, generally done by adding a vinylpyridine or trimethylsilylethyl group to a sulfhydryl; 2) addingadditional subunits to form the conductive oligomer; 3) adding at leasta first component of the redox active complex to the conductiveoligomer; 4) adding additional components as necessary; and 5) attachingthe conductive oligomer to the gold electrode.

The above method may also be used to attach passayation molecules to agold electrode.

In a preferred embodiment, a monolayer of passayation agents is added tothe electrode. Generally, the chemistry of addition is similar to or thesame as the addition of conductive oligomers to the electrode, i.e.using a sulfur atom for attachment to a gold electrode, etc.Compositions comprising monolayers in addition to the conductiveoligomers covalently attached to components of the redox active complexmay be made in at least one of five ways: (1) addition of the monolayer,followed by subsequent addition of the conductive oligomer-redoxactivecomplex; (2) addition of the conductive oligomer-redox activecomplex followed by addition of the monolayer; (3) simultaneous additionof the monolayer and conductive oligomer-redox active complex; (4)formation of a monolayer (using any of 1, 2 or 3) which includesconductive oligomers which terminate in a functional moiety suitable forattachment of a redox active complex; or (5) formation of a monolayerwhich includes conductive oligomers which terminate in a functionalmoiety suitable for synthesis, i.e. the redox active complex (forexample, the binding ligand) is synthesized on the surface of themonolayer as is known in the art. Such suitable functional moietiesinclude, but are not limited to, nucleosides, amino groups, carboxylgroups, protected sulfur moieties, or hydroxyl groups forphosphoramidite additions.

As will be appreciated by those in the art, electrodes may be made thathave any combination of components. Thus, a variety of differentconductive oligomers or passayation agents may be used on a singleelectrode.

Once made, the compositions find use in a number of applications, asdescribed herein.

The compositions of the invention thus comprise assay complexescomprising a target analyte bound to a redox active complex, wherein thecomplex comprises a binding ligand and a redox active molecule. In apreferred embodiment, the ligand-analyte interaction is such that theenvironment of the redox active molecule changes sufficiently uponbinding to alter a measurable redox property of the redox activemolecule. For example, in antibody-antigen complexes, enzyme-substrate(or inhibitor) complexes, other protein-protein interactions, etc., theredox active molecule is generally located within or adjacent to thebinding site or active site of the interaction, such that upon binding,the environment of the redox active molecule changes. This may be due toa conformational change, a “shielding” of the redox active molecule, newsolvent accessibility of the redox active molecule, etc. Preferably, theredox active molecule is placed such that it does not inhibit theligand-target binding but is affected by it. In general, the RAM isgenerally within less than 50 {acute over (Å)} of the target analyte,with less than about 25 {acute over (Å)} being preferred, and less than6-10 {acute over (Å)} being particularly preferred.

In general, changes in faradaic impedance are due to changes in the rateof electron transfer between the RAM, generally through the conductiveoligomer, to the electrode. As predicted by semiclassical theory,changes in the rate of electron transfer can be due to changes in theintervening medium (conceptually, changes in H_(AB)), changes in nuclearreorganization energy, λ (the major component of which is the solventreorganization energy), changes in the driving force (−)G

; which is generally a function of changes in the input signal, ratherthan changes in the system as a result of analyte binding), changes indistance, according to the following equation:k _(ET)=(4B ³ /h ²8k _(B) T)^(1/2)(H _(AB))²exp[(−)G

+8)²/8k _(B) T]

Thus, as generally discussed herein, changes in faradaic impedance aregenerally determined by evaluating the changes in the rate and/orquantity of electron transfer between the RAM and the electrode.Accordingly, changes in faradaic impedance are done by initiatingelectron transfer, generally both in the absence and presence of thetarget analyte, and evaluating the generated signal, which will becharacteristic of either the absence or presence of the target analyte.In some embodiments, for example in system 8, there may be little or noelectron transfer in the absence of the analyte. Other systems rely onchanges in electron transfer rate or quantity on the basis of thepresence or absence of the target analyte.

Electron transfer is generally initiated electronically, with theapplication of at least a first input signal, with voltage beingpreferred. A potential is applied to the assay complex. Precise controland variations in the applied potential can be via a potentiostat andeither a three electrode system (one reference, one sample (or working)and one counter electrode) or a two electrode system (one sample and onecounter electrode). This allows matching of applied potential to peakpotential of the system which depends in part on the choice of RAMs andin part on the conductive oligomer used, the composition and integrityof the monolayer, and what type of reference electrode is used. Asdescribed herein, ferrocene is a preferred RAM.

In a preferred embodiment, a co-reductant or co-oxidant (collectively,co-redoxant) is used, as an additional electron source or sink. Seegenerally Sato et al., Bull. Chem. Soc. Jpn 66:1032 (1993); Uosaki etal., Electrochimica Acta 36:1799 (1991); and Alleman et al., J. Phys.Chem. 100:17050 (1996); all of which are incorporated by reference. Thisfinds use when DC detection modes are used, or slow AC, i.e.non-diffusion limited AC.

In a preferred embodiment, an input electron source in solution is usedin the initiation of electron transfer, preferably when initiation anddetection are being done using DC current or at AC frequencies wherediffusion is not limiting. In general, as will be appreciated by thosein the art, preferred embodiments utilize monolayers that contain aminimum of “holes”, such that short-circuiting of the system is avoided.This may be done in several general ways. In a preferred embodiment, aninput electron source is used that has a lower or similar redoxpotential than the RAM of the assay complex. Thus, at voltages above theredox potential of the input electron source, both the RAM and the inputelectron source are oxidized and can thus donate electrons; the RAMdonates an electron to the electrode and the input source donates to theRAM. For example, ferrocene, as a RAM attached to the compositions ofthe invention as described in the examples, has a redox potential ofroughly 200 mV in aqueous solution (which can change significantlydepending on what the ferrocene is bound to, the manner of the linkageand the presence of any substitution groups). Ferrocyanide, an electronsource, has a redox potential of roughly 200 mV as well (in aqueoussolution). Accordingly, at or above voltages of roughly 200 mV,ferrocene is converted to ferricenium, which then transfers an electronto the electrode. Now the ferricyanide can be oxidized to transfer anelectron to the RAM. In this way, the electron source (or co-reductant)serves to amplify the signal generated in the system, as the electronsource molecules rapidly and repeatedly donate electrons to the RAMattached to the assay complex. The rate of electron donation oracceptance will be limited by the rate of diffusion of the co-reductant,the electron transfer between the co-reductant and the RAM, which inturn is affected by the concentration and size, etc.

Alternatively, input electron sources that have lower redox potentialsthan the RAM are used. At voltages less than the redox potential of theRAM, but higher than the redox potential of the electron source, theinput source such as ferrocyanide is unable to be oxided and thus isunable to donate an electron to the RAM; i.e. no electron transferoccurs. Once ferrocene is oxidized, then there is a pathway for electrontransfer.

In an alternate preferred embodiment, an input electron source is usedthat has a higher redox potential than the RAM of the label probe. Forexample, luminol, an electron source, has a redox potential of roughly720 mV. At voltages higher than the redox potential of the RAM, butlower than the redox potential of the electron source, i.e. 200-720 mV,the ferrocene is oxided, and transfers a single electron to theelectrode via the conductive oligomer. However, the RAM is unable toaccept any electrons from the luminol electron source, since thevoltages are less than the redox potential of the luminol. However, ator above the redox potential of luminol, the luminol then transfers anelectron to the RAM, allowing rapid and repeated electron transfer. Inthis way, the electron source (or co-reductant) serves to amplify thesignal generated in the system, as the electron source molecules rapidlyand repeatedly donate electrons to the RAM of the assay complex.

Luminol has the added benefit of becoming a chemiluminiscent speciesupon oxidation (see Jirka et al., Analytica Chimica Acta 284:345(1993)), thus allowing photo-detection of electron transfer from the RAMto the electrode. Thus, as long as the luminol is unable to contact theelectrode directly, i.e. in the presence of the SAM such that there isno efficient electron transfer pathway to the electrode, luminol canonly be oxidized by transferring an electron to the RAM on the assaycomplex. When the RAM is not present, luminol is not significantlyoxidized, resulting in a low photon emission and thus a low (if any)signal from the luminol. In the presence of the target, a much largersignal is generated. Thus, the measure of luminol oxidation by photonemission is an indirect measurement of the ability of the RAM to donateelectrons to the electrode. Furthermore, since photon detection isgenerally more sensitive than electronic detection, the sensitivity ofthe system may be increased. Initial results suggest that luminescencemay depend on hydrogen peroxide concentration, pH, and luminolconcentration, the latter of which appears to be non-linear.

Suitable electron source molecules are well known in the art, andinclude, but are not limited to, ferricyanide, and luminol.

Alternatively, output electron acceptors or sinks could be used, i.e.the above reactions could be run in reverse, with the RAM such as ametallocene receiving an electron from the electrode, converting it tothe metallicenium, with the output electron acceptor then accepting theelectron rapidly and repeatedly. In this embodiment, cobalticenium isthe preferred RAM.

Changes in the faradaic impedance of the system, e.g. differences in therate or quantity of electron transfer, can be detected in a variety ofways. A variety of detection methods may be used, including, but notlimited to, optical detection (as a result of spectral changes uponchanges in redox states), which includes fluorescence, phosphorescence,luminiscence, chemiluminescence, electrochemiluminescence, andrefractive index; and electronic detection, including, but not limitedto, amperommetry, voltammetry, capacitance and impedence. These methodsinclude time or frequency dependent methods based on AC or DC currents,pulsed methods, lock-in techniques, filtering (high pass, low pass, bandpass), and time-resolved techniques including time-resolvedfluoroscence.

In one embodiment, the efficient transfer of electrons from the RAM tothe electrode results in stereotyped changes in the redox state of theRAM. With many RAMs, including the complexes of ruthenium containingbipyridine, pyridine and imidazole rings, these changes in redox stateare associated with changes in spectral properties. Significantdifferences in absorbance are observed between reduced and oxidizedstates for these molecules. See for example Fabbrizzi et al., Chem. Soc.Rev. 1995 pp 197-202). These differences can be monitored using aspectrophotometer or simple photomultiplier tube device.

In this embodiment, possible electron donors and acceptors include allthe derivatives listed above for photoactivation or initiation.Preferred electron donors and acceptors have characteristically largespectral changes upon oxidation and reduction resulting in highlysensitive monitoring of electron transfer. Such examples includeRu(NH₃)₄py and Ru(bpy)₂im as preferred examples. It should be understoodthat only the donor or acceptor that is being monitored by absorbanceneed have ideal spectral characteristics.

In a preferred embodiment, the electron transfer is detectedfluorometrically. Numerous transition metal complexes, including thoseof ruthenium, have distinct fluorescence properties. Therefore, thechange in redox state of the electron donors and electron acceptorsattached to the redox active complex can be monitored very sensitivelyusing fluorescence, for example with Ru(4,7-biphenyl₂-phenanthroline)₃²⁺. The production of this compound can be easily measured usingstandard fluorescence assay techniques. For example, laser inducedfluorescence can be recorded in a standard single cell fluorimeter, aflow through “on-line” fluorimeter (such as those attached to achromatography system) or a multi-sample “plate-reader” similar to thosemarketed for 96-well immuno assays.

Alternatively, fluorescence can be measured using fiber optic sensorswith binding ligands in solution or attached to the fiber optic.Fluorescence is monitored using a photomultiplier tube or other lightdetection instrument attached to the fiber optic. The advantage of thissystem is the extremely small volumes of sample that can be assayed.

In addition, scanning fluorescence detectors such as the Fluorlmagersold by Molecular Dynamics are ideally suited to monitoring thefluorescence of modified molecules arrayed on solid surfaces. Theadvantage of this system is the large number of electron transfer probesthat can be scanned at once using chips covered with thousands ofdistinct binding ligands.

Many transition metal complexes display fluorescence with large Stokesshifts. Suitable examples include bis- and trisphenanthroline complexesand bis- and trisbipyridyl complexes of transition metals such asruthenium (see Juris, A., Balzani, V., et. al. Coord. Chem. Rev., V. 84,p. 85-277, 1988). Preferred examples display efficient fluorescence(reasonably high quantum yields) as well as low reorganization energies.These include Ru(4,7-biphenyl₂-phenanthroline)₃ ²⁺,Ru(4,4′-diphenyl-2,2′-bipyridine)₃ ²⁺ and platinum complexes (seeCummings et al., J. Am. Chem. Soc. 118:1949-1960 (1996), incorporated byreference). Alternatively, a reduction in fluorescence associated withhybridization can be measured using these systems.

In a further embodiment, electrochemiluminescence is used as the basisof the electron transfer detection. With some RAMs such as Ru²⁺(bpy)₃,direct luminescence accompanies excited state decay. Changes in thisproperty are associated with ligand binding and can be monitored with asimple photomultiplier tube arrangement (see Blackburn, G. F. Clin.Chem. 37: 1534-1539 (1991); and Juris et al., supra.

In a preferred embodiment, electronic detection is used, includingamperommetry, voltammetry, capacitance, and impedence. Suitabletechniques include, but are not limited to, electrogravimetry;coulometry (including controlled potential coulometry and constantcurrent coulometry); voltametry (cyclic voltametry, pulse voltametry(normal pulse voltametry, square wave voltametry, differential pulsevoltametry, Osteryoung square wave voltametry, and coulostatic pulsetechniques); stripping analysis (aniodic stripping analysis, cathiodicstripping analysis, square wave stripping voltammetry); conductancemeasurements (electrolytic conductance, direct analysis); time-dependentelectrochemical analyses (chronoamperometry, chronopotentiometry, cyclicchronopotentiometry and amperometry, AC polography, chronogalvametry,and chronocoulometry); AC impedance measurement; capacitancemeasurement; AC voltametry; and photoelectrochemistry.

In a preferred embodiment, monitoring electron transfer is viaamperometric detection. This method of detection involves applying apotential (as compared to a separate reference electrode) between thecomplex-conjugated electrode and a reference (counter) electrode in thesample containing target genes of interest. Electron transfer ofdiffering efficiencies is induced in samples in the presence or absenceof target analytes; that is, the presence or absence of the targetanalyte can result in different currents.

The device for measuring electron transfer amperometrically involvessensitive current detection and includes a means of controlling thevoltage potential, usually a potentiostat. This voltage is optimizedwith reference to the potential of the electron donating complex on thelabel probe. Possible electron donating complexes include thosepreviously mentioned with complexes of iron, osmium, platinum, cobalt,rhenium and ruthenium being preferred and complexes of iron being mostpreferred.

In a preferred embodiment, alternative electron detection modes areutilized. For example, potentiometric (or voltammetric) measurementsinvolve non-faradaic (no net current flow) processes and are utilizedtraditionally in pH and other ion detectors. Similar sensors are used tomonitor electron transfer between the RAM and the electrode. Inaddition, other properties of insulators (such as resistance) and ofconductors (such as conductivity, impedance and capicitance) could beused to monitor electron transfer between RAM and the electrode.Finally, any system that generates a current (such as electron transfer)also generates a small magnetic field, which may be monitored in someembodiments.

It should be understood that one benefit of the fast rates of electrontransfer observed in the compositions of the invention is that timeresolution can greatly enhance the signal-to-noise results of monitorsbased on absorbance, fluorescence and electronic current. The fast ratesof electron transfer of the present invention result both in highsignals and stereotyped delays between electron transfer initiation andcompletion. By amplifying signals of particular delays, such as throughthe use of pulsed initiation of electron transfer and “lock-in”amplifiers of detection, and Fourier transforms.

In a preferred embodiment, electron transfer is initiated usingalternating current (AC) methods. Without being bound by theory, itappears that RAMs, bound to an electrode, generally respond similarly toan AC voltage across a circuit containing resistors and capacitors.Basically, any methods which enable the determination of the nature ofthese complexes, which act as a resistor and capacitor, can be used asthe basis of detection. Surprisingly, traditional electrochemicaltheory, such as exemplified in Laviron et al., J. Electroanal. Chem.97:135 (1979) and Laviron et al., J. Electroanal. Chem. 105:35 (1979),both of which are incorporated by reference, do not accurately model thesystems described herein, except for very small E_(AC) (less than 10 mV)and relatively large numbers of molecules. That is, the AC current (I)is not accurately described by Laviron's equation. This may be due inpart to the fact that this theory assumes an unlimited source and sinkof electrons, which is not true in the present systems.

Accordingly, alternate equations were developed, using the Nernstequation and first principles to develop a model which more closelysimulates the results. This was derived as follows. The Nernst equation,Equation 1 below, describes the ratio of oxidized (O) to reduced (R)molecules (number of molecules=n) at any given voltage and temperature,since not every molecule gets oxidized at the same oxidation potential.

$\begin{matrix}{{Equation}\mspace{14mu} 1} & \; \\{E_{DC} = {E_{0} + {\frac{RT}{nF}l\; n\frac{\lbrack O\rbrack}{\lbrack R\rbrack}}}} & (1)\end{matrix}$

E_(DC) is the electrode potential, E₀ is the formal potential of themetal complex, R is the gas constant, T is the temperature in degreesKelvin, n is the number of electrons transferred, F is faraday'sconstant, [O] is the concentration of oxidized molecules and [R] is theconcentration of reduced molecules.

The Nernst equation can be rearranged as shown in Equations 2 and 3:

$\begin{matrix}{{Equation}\mspace{14mu} 2} & \; \\{E_{DC} - E_{0} + {\frac{RT}{nF}l\; n\frac{\lbrack O\rbrack}{\lbrack R\rbrack}}} & (2)\end{matrix}$E_(DC) is the DC component of the potential.

$\begin{matrix}{{Equation}\mspace{14mu} 3} & \; \\{\exp^{\frac{nF}{RT}{({E_{DC} - E_{0}})}} = \frac{\lbrack O\rbrack}{\lbrack R\rbrack}} & (3)\end{matrix}$Equation 3 can be rearranged as follows, using normalization of theconcentration to equal 1 for simplicity, as shown in Equations 4, 5 and6. This requires the subsequent multiplication by the total number ofmolecules.[O]+[R]=1  Equation 4[O]=1−[R]  Equation 5[R]=1−[O]  Equation 6

Plugging Equation 5 and 6 into Equation 3, and the fact that nF/RTequals 38.9 V⁻¹, for n=1, gives Equations 7 and 8, which define [O] and[R], respectively:

$\begin{matrix}{{Equation}\mspace{14mu} 7} & \; \\{\lbrack O\rbrack = \frac{\exp^{38.9{({E - E_{0}})}}}{1 + \exp^{38.9{({E - E_{0}})}}}} & (4) \\{{Equation}\mspace{14mu} 8} & \; \\{\lbrack R\rbrack = \frac{1}{1 + \exp^{38.9{({E - E_{0}})}}}} & (5)\end{matrix}$

Taking into consideration the generation of an AC faradaic current, theratio of [O]/[R] at any given potential must be evaluated. At aparticular E_(DC) with an applied E_(AC), as is generally describedherein, at the apex of the E_(AC) more molecules will be in the oxidizedstate, since the voltage on the surface is now (E_(DC)+E_(AC)); at thebottom, more will be reduced since the voltage is lower. Therefore, theAC current at a given E_(DC) will be dictated by both the AC and DCvoltages, as well as the shape of the Nernstian curve. Specifically, ifthe number of oxidized molecules at the bottom of the AC cycle issubtracted from the amount at the top of the AC cycle, the total changein a given AC cycle is obtained, as is generally described by Equation9. Dividing by 2 then gives the AC amplitude.

$\begin{matrix}{{Equation}\mspace{14mu} 9} & \; \\{i_{AC} = \frac{\left( {{electrons}\mspace{14mu}{{at}\left\lbrack {E_{DC} + E_{AC}} \right\rbrack}} \right) - \left( {{electrons}\mspace{14mu}{{at}\left\lbrack {E_{DC} - E_{AC}} \right\rbrack}} \right)}{2}} & \;\end{matrix}$

Equation 10 thus describes the AC current which should result:Equation 10i _(AC) =C ₀ Fω½([O] _(E) _(DC) _(+E) _(AC) −[O] _(E) _(DC) _(−E) _(AC))  (6)

As depicted in Equation 11, the total AC current will be the number ofredox molecules C), times faraday's constant (F), times the AC frequency(ω), times 0.5 (to take into account the AC amplitude), times the ratiosderived above in Equation 7. The AC voltage is approximated by theaverage amplitude, E_(Ac)2/π.

$\begin{matrix}{\mspace{79mu}{{Equation}\mspace{14mu} 11}} & \; \\{i_{AC} = {{\frac{C_{0}F\;\omega}{2}\left( \frac{\exp^{38.9{\lbrack{E_{DC} + \frac{2\; E_{AC}}{\pi} - E_{0}}\rbrack}}}{1 + \exp^{38.9{\lbrack{E + \frac{2\; E_{AC}}{\pi} - E_{0}}\rbrack}}} \right)} - \frac{\exp^{38.9{\lbrack{E_{DC} - \frac{2\; E_{AC}}{\pi} - E_{0}}\rbrack}}}{1 + \exp^{38.9{\lbrack{E_{DC} - \frac{2\; E}{\pi} - E}\rbrack}}}}} & (7)\end{matrix}$

However, Equation 11 does not incorporate the effect of electrontransfer rate nor of instrument factors including input impedance andstray capacitance. Electron transfer rate is important when the rate isclose to or lower than the applied frequency. Thus, the true i_(AC)should be a function of all three, as depicted in Equation 12.i _(AC) =f(Nernst factors)f(k _(ET))f(instrument factors)  Equation 12

These equations can be used to model and predict the expected ACcurrents in systems which use input signals comprising both AC and DCcomponents. As outlined above, traditional theory surprisingly does notmodel these systems at all, except for very low voltages.

In general, non-specifically bound analytes can show differences inimpedance (i.e. higher impedances) than when the analytes arespecifically bound. In a preferred embodiment, the non-specificallybound material is washed away, resulting in an effective impedance ofinfinity. Thus, AC detection gives several advantages as is generallydiscussed below, including an increase in sensitivity, and the abilityto “filter out” background noise. In particular, changes in impedance(including, for example, bulk impedance) as between non-specific bindingof target analytes and target-specific assay complex formation may bemonitored.

Accordingly, when using AC initiation and detection methods, thefrequency response of the system changes as a result of the presence ofthe RAM. By “frequency response” herein is meant a modification ofsignals as a result of electron transfer between the electrode and theRAM. This modification is different depending on signal frequency. Afrequency response includes AC currents at one or more frequencies,phase shifts, DC offset voltages, faradaic impedance, etc.

Once the assay complex including the target analyte is made, a firstinput electrical signal is then applied to the system, preferably via atleast the sample electrode (containing the complexes of the invention)and the counter electrode, to initiate electron transfer between theelectrode and the RAM. Three electrode systems may also be used, withthe voltage applied to the reference and working electrodes. The firstinput signal comprises at least an AC component. The AC component may beof variable amplitude and frequency. Generally, for use in the presentmethods, the AC amplitude ranges from about 1 mV to about 1.1 V, withfrom about 10 mV to about 800 mV being preferred, and from about 10 mVto about 300 mV being especially preferred. The AC frequency ranges fromabout 10 Hz to about 100 KHz, with from about 10 Hz to about 10 MHzbeing preferred, and from about 100 Hz to about 20 MHz being especiallypreferred.

The use of combinations of AC and DC signals gives a variety ofadvantages, including surprising sensitivity and signal maximization.

In a preferred embodiment, the first input signal comprises a DCcomponent and an AC component. That is, a DC offset voltage between thesample and counter electrodes is swept through the electrochemicalpotential of the RAM (for example, when ferrocene is used, the sweep isgenerally from 0 to 500 mV) (or alternatively, the working electrode isgrounded and the reference electrode is swept from 0 to −500 mV). Thesweep is used to identify the DC voltage at which the maximum responseof the system is seen. This is generally at or about the electrochemicalpotential of the RAM. Once this voltage is determined, either a sweep orone or more uniform DC offset voltages may be used. DC offset voltagesof from about −1 V to about +1.1 V are preferred, with from about −500mV to about +800 mV being especially preferred, and from about −300 mVto about 500 mV being particularly preferred. In a preferred embodiment,the DC offset voltage is not zero. On top of the DC offset voltage, anAC signal component of variable amplitude and frequency is applied. Ifthe RAM is present, and can respond to the AC perturbation, an ACcurrent will be produced due to electron transfer between the electrodeand the RAM.

For defined systems, it may be sufficient to apply a single input signalto differentiate between the presence and absence of the target analyte.Alternatively, a plurality of input signals are applied. As outlinedherein, this may take a variety of forms, including using multiplefrequencies, multiple DC offset voltages, or multiple AC amplitudes, orcombinations of any or all of these.

Thus, in a preferred embodiment, multiple DC offset voltages are used,although as outlined above, DC voltage sweeps are preferred. This may bedone at a single frequency, or at two or more frequencies.

In a preferred embodiment, the AC amplitude is varied. Without beingbound by theory, it appears that increasing the amplitude increases thedriving force. Thus, higher amplitudes, which result in higheroverpotentials give faster rates of electron transfer. Thus, generally,the same system gives an improved response (i.e. higher output signals)at any single frequency through the use of higher overpotentials at thatfrequency. Thus, the amplitude may be increased at high frequencies toincrease the rate of electron transfer through the system, resulting ingreater sensitivity. In addition, this may be used, for example, toinduce responses in slower systems such as those that do not possessoptimal spacing configurations.

In a preferred embodiment, measurements of the system are taken at leasttwo separate amplitudes or overpotentials, with measurements at aplurality of amplitudes being preferred. As noted above, changes inresponse as a result of changes in amplitude may form the basis ofidentification, calibration and quantification of the system. Inaddition, one or more AC frequencies can be used as well.

In a preferred embodiment, the AC frequency is varied. At differentfrequencies, different molecules respond in different ways. As will beappreciated by those in the art, increasing the frequency generallyincreases the output current. However, when the frequency is greaterthan the rate at which electrons may travel between the electrode andthe RAM higher frequencies result in a loss or decrease of outputsignal. At some point, the frequency will be greater than the rate ofelectron transfer between the RAM and the electrode, and then the outputsignal will also drop.

In one embodiment, detection utilizes a single measurement of outputsignal at a single frequency. That is, the frequency response of thesystem in the absence of target analyte can be previously determined tobe very low at a particular high frequency. Using this information, anyresponse at a particular frequency, will show the presence of the assaycomplex. That is, any response at a particular frequency ischaracteristic of the assay complex. Thus, it may only be necessary touse a single input high frequency, and any changes in frequency responseis an indication that the analyte is present, and thus that the targetsequence is present.

In addition, the use of AC techniques allows the significant reductionof background signals at any single frequency due to entities other thanthe analytes, i.e. “locking out” or “filtering” unwanted signals. Thatis, the frequency response of a charge carrier or redox active moleculein solution will be limited by its diffusion coefficient and chargetransfer coefficient. Accordingly, at high frequencies, a charge carriermay not diffuse rapidly enough to transfer its charge to the electrode,and/or the charge transfer kinetics may not be fast enough. This isparticularly significant in embodiments that do not have goodmonolayers, i.e. have partial or insufficient monolayers, i.e. where thesolvent is accessible to the electrode. As outlined above, in DCtechniques, the presence of “holes” where the electrode is accessible tothe solvent can result in solvent charge carriers “short circuiting” thesystem, i.e. the reach the electrode and generate background signal.However, using the present AC techniques, one or more frequencies can bechosen that prevent a frequency response of one or more charge carriersin solution, whether or not a monolayer is present. This is particularlysignificant since many biological fluids such as blood containsignificant amounts of redox active molecules which can interfere withamperometric detection methods.

In a preferred embodiment, measurements of the system are taken at atleast two separate frequencies, with measurements at a plurality offrequencies being preferred. A plurality of frequencies includes a scan.For example, measuring the output signal, e.g., the AC current, at a lowinput frequency such as 1-20 Hz, and comparing the response to theoutput signal at high frequency such as 10-100 kHz will show a frequencyresponse difference between the presence and absence of the RAM. In apreferred embodiment, the frequency response is determined at at leasttwo, preferably at least about five, and more preferably at least aboutten frequencies.

After transmitting the input signal to initiate electron transfer, anoutput signal is received or detected. The presence and magnitude of theoutput signal will depend on a number of factors, including theoverpotential/amplitude of the input signal; the frequency of the inputAC signal; the composition of the intervening medium; the DC offset; theenvironment of the system; the nature of the RAM; the solvent; and thetype and concentration of salt. At a given input signal, the presenceand magnitude of the output signal will depend in general on thepresence or absence of the target analyte, the placement and distance ofthe RAM from the surface of the monolayer and the character of the inputsignal. In some embodiments, it may be possible to distinguish betweennon-specific binding of materials and the formation of target specificassay complexes, on the basis of impedance.

In a preferred embodiment, the output signal comprises an AC current. Asoutlined above, the magnitude of the output current will depend on anumber of parameters. By varying these parameters, the system may beoptimized in a number of ways.

In general, AC currents generated in the present invention range fromabout 1 femptoamp to about 1 milliamp, with currents from about 50femptoamps to about 100 microamps being preferred, and from about 1picoamp to about 1 microamp being especially preferred.

In a preferred embodiment, the output signal is phase shifted in the ACcomponent relative to the input signal. Without being bound by theory,it appears that the systems of the present invention may be sufficientlyuniform to allow phase-shifting based detection. That is, the complexbiomolecules of the invention through which electron transfer occursreact to the AC input in a homogeneous manner, similar to standardelectronic components, such that a phase shift can be determined. Thismay serve as the basis of detection between the presence and absence ofthe analyte and/or differences between the presence of target-specificassay complexes and non-specific binding of materials to the systemcomponents.

The output signal is characteristic of the presence of the analyte; thatis, the output signal is characteristic of the presence of thetarget-specific assay complex. In a preferred embodiment, the basis ofthe detection is a difference in the faradaic impedance of the system asa result of the formation of the assay complex. Of importance in themethods of the invention is that the faradaic impedance between the RAMand the electrode may be significantly different depending on whetherthe targets are specifically or non-specifically bound to the electrode.

Accordingly, the present invention further provides electronic devicesor apparatus for the detection of analytes using the compositions of theinvention. The apparatus includes a test chamber for receiving a samplesolution which has at least a first measuring or sample electrode, and asecond measuring or counter electrode. Three electrode systems are alsouseful. The first and second measuring electrodes are in contact with atest sample receiving region, such that in the presence of a liquid testsample, the two electrodes may be in electrical contact.

In a preferred embodiment, the apparatus also includes detectionelectrodes comprising the compositions of the invention, including redoxactive complexes including binding ligands and redox active molecules,and a monolayer comprising conductive oligomers, such as are describedherein.

The apparatus further comprises an AC voltage source electricallyconnected to the test chamber; that is, to the measuring electrodes.Preferably, the AC voltage source is capable of delivering DC offsetvoltage as well.

In a preferred embodiment, the apparatus further comprises a processorcapable of comparing the input signal and the output signal. Theprocessor is coupled to the electrodes and configured to receive anoutput signal, and thus detect the presence of the target.

Thus, the compositions of the present invention may be used in a varietyof research, clinical, quality control, or field testing settings.

In a preferred embodiment, viral and bacterial detection is done usingthe complexes of the invention. In this embodiment, binding ligands (forexample antibodies or fragments thereof) are designed to detect targets(for example surface proteins) from a variety of bacteria and viruses.For example, current blood-screening techniques rely on the detection ofanti-HIV antibodies. The methods disclosed herein allow for directmonitoring of circulating virus within a patient as an improved methodof assessing the efficacy of anti-viral therapies. Similarly, virusesassociated with leukemia, HTLV-I and HTLV-II, may be detected in thisway. Bacterial infections such as tuberculosis, clymidia and othersexually transmitted diseases, may also be detected. Similarly, thecompositions of the invention find use as probes for toxic bacteria inthe screening of water and food samples. Similarly, bioremediationstrategies may be evaluated using the compositions of the invention.

The present invention provides methods which can result in sensitivedetection of target analytes. In a preferred embodiment, less than about10¹² molecules are detected, with less than about 10¹⁰ being preferred,less than 10⁸ being particularly preferred, less than about 10⁵ beingespecially preferred, and less than about 10⁴ being most preferred.

All references cited herein are incorporated by reference in theirentirety.

1. A method of detecting an enzyme protein target analyte in a samplecomprising: a) applying a first input signal to an assay complexcomprising: i) a redox active complex comprising: 1) a redox activemolecule; 2) an enzyme substrate that will interact with the enzymeprotein target analyte; and ii) said enzyme protein target analyte,wherein said enzyme substrate is covalently attached to a firstmeasuring electrode via a spacer, and wherein said first measuringelectrode comprises: a passivation agent monolayer comprising at least acovalently attached first passivation species and a covalently attachedsecond passivation species comprising said enzyme substrate; and b)detecting a change in the faradaic impedance of the system as a resultof the interaction of the enzyme substrate with said enzyme proteintarget analyte, if present.
 2. The method according to claim 1, whereinsaid input signal comprises an AC component.
 3. The method according toclaim 1, wherein said input signal further comprises a DC component. 4.The method according to claim 1, wherein said redox active molecule iscovalently attached to said electrode.
 5. The method according to claim1, wherein said enzyme substrate is covalently attached to saidelectrode.
 6. The method according to claim 1, wherein said redox activemolecule is a transition metal complex.
 7. The method according to claim6, wherein said transition metal complex is ferrocene.
 8. The methodaccording to claim 1, wherein said redox active molecule is covalentlyattached to said enzyme substrate.
 9. The method according to claim 1,wherein said detecting is by receiving an output signal characteristicof the presence of said analyte.
 10. The method according to claim 9,wherein said output signal comprises a current.
 11. The method accordingto claim 1, wherein said enzyme substrate comprises a protein.
 12. Themethod according to claim 11 wherein said protein is a peptide.
 13. Themethod according to claim 1, wherein said passivation agent monolayercomprises insulators.
 14. The method according to claim 1, wherein saidpassivation agent monolayer comprises conductive oligomers.
 15. Themethod according to claim 1, wherein said passivation agent monolayercomprises insulators and conductive oligomers.
 16. The method accordingto claim 15, wherein said insulator comprises a C1 to C12 alkyl group.17. The method according to claim 1, wherein enzyme substrate iscovalently attached to said redox active molecule to form a redox activecomplex.
 18. The method according to claim 17, wherein an atom of saidenzyme substrate serves as a coordination atom to the transition metalto form a redox active complex.
 19. The method according to claim 17,wherein said covalent attachment utilizes more than one sulfur moiety.20. The method according to claim 1, wherein said redox active moleculeand said enzyme substrate are covalently attached to said firstmeasuring electrode separately.